Denitrifying Genes Research Articles

Manure application increased denitrifying gene abundance in a drip-irrigated cotton field.

Application of inorganic nitrogen (N) fertilizer and manure can increase nitrous oxide (N2O) emissions. We tested the hypothesis that increased N2O flux from soils amended with manure reflects a change in bacterial community structure and, specifically, an increase in the number of denitrifiers. To test this hypothesis, a field experiment was conducted in a drip-irrigated cotton field in an arid region of northwestern China. Treatments included plots that were not amended (Control), and plots amended with urea (Urea), animal manure (Manure) and a 50/50 mix of urea and manure (U+M). Manure was broadcast-incorporated into the soil before seeding while urea was split-applied with drip irrigation (fertigation) over the growing season. The addition treatments did not, as assessed by nextgen sequencing of PCR-amplicons generated from rRNA genes in soil, affect the alpha diversity of bacterial communities but did change the beta diversity. Compared to the Control, the addition of manure (U+M and Manure) significantly increased the abundance of genes associated with nitrate reduction (narG) and denitrfication (nirK and nosZ). Manure addition (U+M and Manure) did not affect the nitrifying enzyme activity (NEA) of soil but resulted in 39–59 times greater denitrifying enzyme activity (DEA). In contrast, urea application had no impact on the abundances of nitrifier and denitrifier genes, DEA and NEA; likely due to a limitation of C availability. DEA was highly correlated (r = 0.70–0.84, P < 0.01) with the abundance of genes narG, nirK and nosZ. An increase in the abundance of these functional genes was further correlated with soil NO3−, dissolved organic carbon, total C, and total N concentrations, and soil C:N ratio. These results demonstrated a positive relationship between the abundances of denitrifying functional genes (narG, nirK and nosZ) and denitrification potential, suggesting that manure application increased N2O emission by increasing denitrification and the population of bacteria that mediated that process.

Presence of spring-thaw N2O emissions are not linked to functional gene abundance in a drip-fertigated cropped soil in arid northwestern China

Spring-thaw represents a significant source for nitrous oxide (N2O) emissions from fertilized croplands in temperate regions. In this study, we present surface N2O fluxes, soil-profile N2O concentrations at 5, 15, 30 and 60 cm depths along with the abundance of nitrifiers and denitrifiers over the winter and spring-thaw periods in an arid, drip- fertigated cotton field, which had received spring application of 240 kg N ha−1 as urea alone (Urea), polymer-coated urea (ESN), and urea plus urease and nitrification inhibitors. Nitrous oxide emissions from December to April were generally unaffected by fertilizer treatments with a cumulative average of 186 g N ha−1, accounting for 39% of the annual N2O emissions. Emission peaks occurred at spring-thaw and coincided with increasing soil-profile N2O concentrations at all depths, suggesting the burst in N2O fluxes was due to new N2O production, rather than a physical release of N2O trapped in the soil profiles over winter. The abundance of nitrifier and denitrifier genes changed over the winter and spring-thaw periods but was not affected by fertilizer treatments from the previous spring, suggesting the abundance of N2O-producing microorganism was more controlled by environmental conditions than N sources applied in the previous spring. The daily N2O flux rate from December to April was positively correlated with soil temperature, water-filled pore space, and denitrifying enzyme activity, but not with the gene copy number of AOA, AOB, narG, nirS, nirK and nosZ, indicating that variation in the abundance of these genes was not contributing to the N2O emissions. These results suggest that N2O emissions in spring-thaw are substantial for drip-fertigated croplands in the arid regions and should be considered in the annual budgets. The environmental factors such as soil temperature and moisture are likely more important than the copy-numbers of N2O-producing functional genes in driving the variability in spring-thaw emissions.

Mining the Drilosphere: Bacterial Communities and Denitrifier Abundance in a No-Till Wheat Cropping System.

Earthworms play important roles in no-till cropping systems by redistributing crop residue to lower soil horizons, providing macropores for root growth, increasing water infiltration, enhancing soil quality and organic matter, and stimulating nitrogen cycling. The soil impacted by earthworm activity, including burrows, casts, and middens, is termed the drilosphere. The objective of this study was to determine the effect of earthworms on soil microbial community composition in the drilosphere at different landscape slope positions. Soil cores (50 cm depth) were extracted from three landscape locations (top, middle, and bottom slope positions) on a sloping aspect of a no-till wheat farm. Soil was sampled at the bottom of the soil core from inside multiple earthworm (Lumbricus terrestris) channels (drilosphere) and from adjacent bulk soil. Bacterial communities were characterized for 16S rRNA gene diversity using high-throughput sequencing and functional denitrifier gene abundance (nirK, nirS, and nosZ) by quantitative PCR. Bacterial communities were structured primarily by the landscape slope position of the soil core followed by source (bulk versus drilosphere soil), with a significant interaction between core position and source. The families AKIW874, Chitinophagaceae, and Comamonadaceae and the genera Amycolatopsis, Caulobacter, Nocardioides, and Variovorax were more abundant in the drilosphere compared to the bulk soil. Most of the individual bacterial taxa enriched in the drilosphere versus bulk soil were members of Actinobacteria, including Micrococcales, Gaiellaceae, Solirubrobacterales, and Mycobacterium. In general, the greatest differences in communities were observed in comparisons of the top and bottom slope positions in which the bottom slope communities had significantly greater richness, diversity, and denitrifier abundance than the top slope position. Populations of denitrifiers (i.e., ratio of nirK+nirS to 16S rRNA) were more abundant in earthworm-impacted soils and there was a significant impact of L. terrestris on soil community composition which was observed only in the top landscape position. There were significant correlations between the abundance of nirK and nirS and taxa within Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia, and Chloroflexi, suggesting a broad diversity of denitrifying bacteria. Earthworms influence the soil microbial communities, but the impact depends on the slope location in a variable landscape, which likely reflects different soil characteristics.

Comparative study on nitrogen removal and functional genes response between surface flow constructed wetland and floating treatment wetland planted with Iris pseudacorus.

Excessive nitrogen accumulated from wastewater with low C/N ratio is a new threat to water ecosystem. In this study, surface flow constructed wetland (SFCW) and floating treatment wetland (FTW) planted with Iris pseudacorus were set in parallel for nitrogen removal. The nitrogen removal efficiencies and pathways, as well as the abundance and functional diversities of the microbial community, were investigated. The results demonstrated that SFCW generally had better nitrogen removal performance than FTW did over four seasons. The average total nitrogen removal efficiency was 66.0% and 43.8% in SFCW and FTW, respectively. The plant uptake played a vital role in nitrogen reduction, which accounted for 29.3% and 7.7% of the total removed nitrogen in SFCW and FTW, respectively. A combination of high-throughput sequencing and quantitative polymerase chain reaction analysis revealed that the two wetland systems had complete nitrogen cycling, and the narG gene was the dominant nitrogen-transformation functional gene in both systems. More abundant denitrifying genes in SFCW than in FTW were also responsible for higher removal capacity of nitrogen. The results suggest that the planting pattern of wetland vegetation has an important impact on nitrogen removal efficiency by influencing the plant absorption and the development of microbial communities.

Responses of nitrification and denitrification to nitrogen and phosphorus fertilization: does the intrinsic soil fertility matter?

Nitrogen (N) is not only a major regulator of productivity in terrestrial systems but can also be a pollutant. While the effects of fertilizer addition to soil N cycling processes and the associated soil microbial groups have been extensively studied, little attention has been paid to the interaction of fertilizer amendment and the intrinsic fertility of the soil. We conducted a filed study on the Chinese Loess Plateau with soils of two fertility levels (low fertility and high fertility), amended with or without synthetic fertilizer (N and phosphorus, NP). Soil samples were collected three times in a continuous winter wheat cropping system and analyzed for soil potential nitrification (PNA) and denitrification (DEA) rates, nitrifiers and denitrifiers. Populations of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and the resulting PNA were increased by the NP application regardless of intrinsic soil fertility. The NP application increased DEA by 15–228% in the low fertility soil, but decreased DEA by 18–46% in the high fertility soil. Soil denitrifiers also showed divergent responses to NP application in low and high fertility soils, i.e., the abundance of nirS-nitrite reducers increased in the low fertility soil, while nirS- and nirK-nitrite reducers were unchanged in the high fertility soil. The narG-nitrate reducers, AOB and AOA were more responsive to the NP application in the tillering stage (BBCH 25–26) than in other periods, whereas PNA, DEA and denitrifier gene abundance (nirS and nirK) varied more in the flowering stage (BBCH 65). Our results suggest that the responses of soil nitrification/nitrifiers and denitrification /denitrifiers to NP application varied with intrinsic soil fertility levels. The contrasting responses of nitrification and denitrification to NP application in high fertility soil indicate a decoupling of both processes.

Vegetation Effects on Bacteria and Denitrifier Abundance in the Soils of Two Tidal Freshwater Wetlands in Virginia

This study examined the abundance of bacteria and nirS-type denitrifiers associated with the rhizospheres of three emergent macrophyte species (Juncus effusus, Typha latifolia, and Peltandra virginica) to gain a greater understanding of plant-microbe interactions in wetland soils. Sampling of plant and soil properties was performed during the growing season (June) and following plant senescence (November) at two tidal freshwater wetlands. Quantitative polymerase chain reaction was used to determine the abundance of bacteria (16S rRNA) and nirS-type denitrifier genes from the rhizosphere and rhizoplane of each plant species and from nearby unvegetated soils. For bacteria, there was a positive rhizosphere effect that did not differ significantly across plant species. In contrast, significant differences in the abundance of nirS-type denitrifiers were observed across the plant species. Rhizosphere abundance was ∼2-fold greater in Peltandra virginica and 4-fold greater in Typha latifolia compared to Juncus effusus. For both bacteria and nirS-type denitrifiers, plant effects were greater during the growing season, and abundance was highly correlated with soil pH, moisture, and organic matter content. Overall, these results demonstrate plant effects on the rhizosphere microbial community can be species‐specific and that there is a synergistic relationship between plant species and environmental conditions.

Effects of carbon source, C/N ratio, nitrate, temperature, and pH on N2O emission and functional denitrifying genes during heterotrophic denitrification

The effects of operational parameters such as carbon source, C/N ratio, initial nitrate concentration, temperature, and pH value on heterotrophic denitrification and functional denitrifying genes were evaluated. When methanol was used as the sole carbon source, complete denitrification was performed in a short time without nitrous oxide (N2O) emission. Complete denitrification was performed at high C/N ratios (5.14 and 12.85) and low initial nitrate concentrations (75.9 and 151.6 mg N L−1). The denitrification rate was not temperature-sensitive in the range of 25–35 °C, but tended to decrease at a low pH of 5–6. The relationships between N2O emission and functional genes under various operational conditions were investigated by Pearson correlation and association network analyses. The C/N ratio was a key factor for N2O emission during the heterotrophic denitrification process. This information on the denitrification performance and its association with functional gene dynamics under various operational conditions is useful for N2O mitigation strategies for wastewater treatment processes.

Impacts of surface-applied residues on N-cycling soil microbial communities in miscanthus and switchgrass cropping systems

The production of biomass crops such as the perennial grasses (PGs) miscanthus (Miscanthus spp.) and switchgrass (Panicum virgatum L.) has increased considerably. The repeated annual harvest of aboveground PG biomass removes organic inputs from the soil and may influence soil health and soil microbial communities, which drive terrestrial nitrogen (N) cycling, influencing the ecosystem services provided by these feedstock systems. Our objective was to assess soil bacterial N-cycling communities as influenced by the return or removal of aboveground plant biomass (residues) to soils in PG biomass feedstock systems at different N fertilization (0 or 160 kg N ha−1) rates. Soil was collected from a field trial and quantitative PCR was used to enumerate bacterial 16S rRNA and denitrifying (nirS and nosZ) genes and transcripts. Denitrifier gene expression (nirS and nosZ) was significantly higher in N-fertilized compared to unfertilized plots, indicating that applying fertilizer in these systems may shift the activity of the denitrifying populations and possibly lead to associated N losses with no return in yield. Returning biomass residues resulted in significantly higher nosZ transcripts than in soils with residues removed but did not influence nirS gene expression. The removal of plant residues in these systems may influence the activity of the nitrous-oxide reducing microbial community, resulting in potential changes in the ecosystem services moderated by soil microbial communities, which may need to be incorporated into future soil health assessments of bioenergy feedstock systems.

Nitrosospira sp. Govern Nitrous Oxide Emissions in a Tropical Soil Amended With Residues of Bioenergy Crop.

Organic vinasse, a residue produced during bioethanol production, increases nitrous oxide (N2O) emissions when applied with inorganic nitrogen (N) fertilizer in soil. The present study investigated the role of the ammonia-oxidizing bacteria (AOB) community on the N2O emissions in soils amended with organic vinasse (CV: concentrated and V: non-concentrated) plus inorganic N fertilizer. Soil samples and N2O emissions were evaluated at 11, 19, and 45 days after fertilizer application, and the bacterial and archaea gene (amoA) encoding the ammonia monooxygenase enzyme, bacterial denitrifier (nirK, nirS, and nosZ) genes and total bacteria were quantified by real time PCR. We also employed a deep amoA amplicon sequencing approach to evaluate the effect of treatment on the community structure and diversity of the soil AOB community. Both vinasse types applied with inorganic N application increased the total N2O emissions and the abundance of AOB. Nitrosospira sp. was the dominant AOB in the soil and was correlated with N2O emissions. However, the diversity and the community structure of AOB did not change with vinasse and inorganic N fertilizer amendment. The results highlight the importance of residues and fertilizer management in sustainable agriculture and can be used as a reference and an input tool to determine good management practices for organic fertilization.

How shallow water table conditions affect N2O emissions and associated microbial abundances under different nitrogen fertilisations

Globally, agriculture is the largest source of nitrous oxide (N2O), a potent greenhouse gas (GHG). A recognised tool to prevent its loss from agricultural soils is the presence of a shallow water table. A four-year lysimeter experiment (2011–2014) was conducted in northeast Italy to investigate how water table levels affect N2O emissions after different N fertilisation techniques. Soil surface flux and groundwater-dissolved N2O were studied under free drainage and at two shallow water table levels (60 cm and 120 cm) and at two levels of N input (250 and 368 kg N ha−1 y−1), using dry manure in 2011 and 2012 and fresh manure in 2013 and 2014. DNA was extracted from soils and quantitative PCR (qPCR) was used to assess the size of nitrifying, denitrifying and N2-fixing bacterial communities. at three soil depths. The day after pre-seeding fertiliser incorporation, N2O emission started to be detected and continued for two-three weeks; brief measurable emissions also followed top–dressing fertilisation events. Cumulative N2O emission measured between 0.97 and 2.33 kg N2O-N ha−1 y−1, corresponding to emission factors from 0.4% to 1.1%. Manure fertilisation significantly affected the N dose only when applied as fresh manure. Water-filled pore space (WFPS) affected daily N2O emissions with a significant interaction with fertilisation level. The two N input levels showed differences only when WFPS was >40%, which revealed N availability as key to increased N2O emissions at high water content, supposedly by fostering anaerobic denitrification. No significant relationships were observed between peak N2O emissions and the values of the temperature or irrigation variables recorded during the experimental observation period. Groundwater dissolved N2O-N concentrations measured about 1.7 μg L−1 with some peak variability from nitrate leaching. Quantitative PCR assays demonstrated that shifts in microbial population that can be involved in oxidation processes and heterotrophic denitrification occurred in the soil, even though the contributions of the different N pathways on N2O emissions were indistinguishable. Indeed, both nitrifying and denitrifying genes were simultaneously promoted by the high fertilisation input and hindered by the high water table level. Shallow groundwater conditions appeared to reduce N2O emissions probably by favouring complete denitrification. These results suggest that in the Po Plain, regulated by the Nitrate Directive, shallow groundwater conditions, with a balanced N input, may mitigate air and water pollution.

Reactor staging influences microbial community composition and diversity of denitrifying MBBRs- Implications on pharmaceutical removal

The subdivision of biofilm reactor in two or more stages (i.e., reactor staging) represents an option for process optimisation of biological treatment. In our previous work, we showed that the gradient of influent organic substrate availability (induced by the staging) can influence the microbial activity (i.e., denitrification and pharmaceutical biotransformation kinetics) of a denitrifying three-stage Moving Bed Biofilm Reactor (MBBR) system. However, it is unclear whether staging and thus the long-term exposure to varying organic carbon type and loading influences the microbial community structure and diversity. In this study, we investigated biofilm structure and diversity in the three-stage MBBR system (S) compared to a single-stage configuration (U) and their relationship with microbial functions. Results from 16S rRNA amplicon libraries revealed a significantly higher microbial richness in the staged MBBR (at 99% sequence similarity) compared to single-stage MBBR. A more even and diverse microbial community was selected in the last stage of S (S3), likely due to exposure to carbon limitation during continuous-flow operation. A core of OTUs was shared in both systems, consisting of Burkholderiales, Xanthomonadales, Flavobacteriales and Sphingobacteriales, while MBBR staging selected for specific taxa (i.e., Candidate division WS6 and Deinococcales). Results from quantitative PCR (qPCR) showed that S3 exhibited the lowest abundance of 16S rRNA but the highest abundance of atypical nosZ, suggesting a selection of microbes with more diverse N-metabolism (i.e., incomplete denitrifiers) in the stage exposed to the lowest carbon availability. A positive correlation (p < 0.05) was observed between removal rate constants of several pharmaceuticals with abundance of relevant denitrifying genes, but not with biodiversity. Despite the previously suggested positive relationship between microbial diversity and functionality in macrobial and microbial ecosystems, this was not observed in the current study, indicating a need to further investigate structure-function relationships for denitrifying systems.

Effect of model root exudate on denitrifier community dynamics and activity at different water-filled pore space levels in a fertilised soil

Although a “rhizosphere effect” on denitrification rates has been established, a clear understanding of the effects of exudate addition on denitrifier community dynamics remains elusive. A microcosm experiment was designed to explore the interaction between exudate addition and soil moisture on community dynamics and denitrification rates. Artificial root exudate at 5 different carbon concentrations was added daily to soil microcosms at contrasting target WFPS (50, 70 and 90%). After a 7-day period, total denitrification and N2O emission rates were measured and community dynamics assessed using molecular methods. The response of denitrifier genes to exudate addition was different, with nirS and nosZ-I showing a stronger effect than nirK and nosZ-II. Distinct community structures were observed for nirS and nosZ-I at 90% target WFPS when compared to 50% and 70%. NirS denitrifier population size showed a ca. 5-Fold increase in gene copy number at 90% WFPS when exudate was added at the highest C input. Significant total denitrification and N2O emission rates were observed only at 90% WFPS, which increased with C input. Our study improves the understanding of the complex interaction between microbial communities, the abiotic environment and process rates which can inform management practices aimed at increasing complete denitrification and controlling greenhouse gas production from agriculture.

Isotope and microbiome data provide complementary information to identify natural nitrate attenuation processes in groundwater

Natural attenuation processes alleviate the impact of fertilization practices on groundwater resources. Therefore, identifying the occurrence of denitrification has become a requirement for water quality management. Several approaches are useful for this purpose, such as isotopic and microbiological methods, each of them providing distinct but complementary information about denitrification reactions, attenuation rates and their occurrence in the aquifer. In this paper, we investigate the contribution of both approaches to describe denitrification in a consolidated rock aquifer (limestone and marls), with a porosity related to fracture networks located in the northeastern sector of the Osona basin (NE Spain).Isotopic methods indicated the origin of nitrate (fertilization using manure) and that denitrification occurred, reaching a reduction of near 25% of the nitrate mass in groundwater. The studied area could be divided in two zones with distinct agricultural pressures and, consequently, nitrate concentrations in groundwater. Denitrification occurred in both zones and at different levels, indicating that attenuation processes took place all along the whole hydrogeological unit, and that the observed levels could be attributed to a larger flow path or, in a minor extent, to mixing processes that mask the actual denitrification rates.Microbiological data showed a correlation between denitrifier genes and the isotopic composition. However, the groundwater microbiome and the distribution of denitrifying bacteria did not reveal a major influence on the denitrification level observed by isotopic methods. This focuses the interest of microbiological analysis to identify functional genes within the bacteria present in the aquifer.Results indicated that isotopic methods provide information of the overall denitrification ability of the hydrogeological unit, and that genomic data represent the processes actually acting nearby the well. A combination of both approaches is advised to support induced in situ attenuation actions in polluted sites.

Effect of P stoichiometry on the abundance of nitrogen-cycle genes in phosphorus-limited paddy soil

Previous studies have shown that phosphorus addition to P-limited soils increases gaseous N loss. A possible explanation for this phenomenon is element stoichiometry (specifically of C:N:P) modifying linked nutrient cycling, leading to enhanced nitrification and denitrification. In this study, we investigated how P stoichiometry influenced the dynamics of soil N-cycle functional genes. Rice seedlings were planted in P-poor soils and incubated with or without P application. Quantitative PCR was then applied to analyze the abundance of ammonia-oxidizing (amoA) and denitrifying (narG nirK, nirS, nosZ) genes in soil. P addition reduced bacterial amoA abundance but increased denitrifying gene abundance. We suggest this outcome is due to P-induced shifts in soil C:P and N:P ratios that limited ammonia oxidization while enhancing P availability for denitrification. Under P application, the rhizosphere effect raised ammonia-oxidizing bacterial abundance (amoA gene) and reduced nirK, nirS, and nosZ in rhizosphere soils. The change likely occurred through greater C input and O2 release from roots, thus altering C availability and redox conditions for microbes. Our results show that P application enhances gaseous N loss potential in paddy fields mainly through stimulating denitrifier growth. We conclude that nutrient availability and elemental stoichiometry are important in regulating microbial gene responses, thereby influencing key ecosystem processes such as denitrification.

Soil nitrogen transformation responses to seasonal precipitation changes are regulated by changes in functional microbial abundance in a subtropical forest

Abstract. The frequency of dry-season droughts and wet-season storms has been predicted to increase in subtropical areas in the coming decades. Since subtropical forest soils are significant sources of N2O and NO3−, it is important to understand the features and determinants of N transformation responses to the predicted precipitation changes. A precipitation manipulation field experiment was conducted in a subtropical forest to reduce dry-season precipitation and increase wet-season precipitation, with annual precipitation unchanged. Net N mineralization, net nitrification, N2O emission, nitrifying (bacterial and archaeal amoA) and denitrifying (nirK, nirS and nosZ) gene abundance, microbial biomass carbon (MBC), extractable organic carbon (EOC), NO3−, NH4+ and soil water content (SWC) were monitored to characterize and explain soil N transformation responses. Dry-season precipitation reduction decreased net nitrification and N mineralization rates by 13–20 %, while wet-season precipitation addition increased both rates by 50 %. More than 20 % of the total variation of net nitrification and N mineralization could be explained by microbial abundance and SWC. Notably, archaeal amoA abundance showed the strongest correlation with net N transformation rates (r ≥ 0.35), suggesting the critical role of archaeal amoA abundance in determining N transformations. Increased net nitrification in the wet season, together with large precipitation events, caused substantial NO3− losses via leaching. However, N2O emission decreased moderately in both dry and wet seasons due to changes in nosZ gene abundance, MBC, net nitrification and SWC (decreased by 10–21 %). We conclude that reducing dry-season precipitation and increasing wet-season precipitation affect soil N transformations through altering functional microbial abundance and MBC, which are further affected by changes in EOC and NH4+ availabilities.

Soil water content modulates the effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on nitrifying and denitrifying bacteria

In the last decades, intensive agriculture management and the application of nitrogen fertilizer have led to high nitrogen losses to the environment. Among these losses is the soil emission of the potent greenhouse gas nitrous oxide (N2O) which is mainly produced by the soil microbial processes of nitrification and denitrification. The use of nitrification inhibitors with ammonium based fertilizers can be an efficient mitigation strategy to decrease these nitrogen losses. Soil water content and temperature are major factors controlling N2O production and, so, also the efficiency of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) to reduce these emissions. In this work we study the effect of soil water content and temperature on the abundance of soil bacteria, ammonia-oxidizing bacteria and denitrifying bacteria, and on the performance of DMPP modulating these different bacterial populations responsible for N2O production/consumption. The effect of DMPP on the abundance of the different bacterial populations was dependent on soil water content. Under 40% of soil water filled pore space (WFPS), the application of DMPP caused an expected decrease of the target amoA gene abundance while narG, nirK and nosZ gene abundances also decreased probably due to an indirect effect induced by the lack of substrate for denitrifiers. Under 80% of WFPS, while the application of DMPP did not induce any change in the amoA gene abundance, an unexpected increase in non-target denitrifying gene abundances (narG, nirK and nosZ) was observed at either 10°C or 20°C incubation temperatures. This finding opens a new gate of research in the use of DMPP, where nitrous oxide reductase enzyme gene (nosZ) abundance and expression should be studied in the future in order to better understand the performance of DMPP not only inhibiting nitrification, but also affecting the non-target denitrification process.

Effects of root exudates on denitrifier gene abundance, community structure and activity in a micro-polluted constructed wetland

In micro-polluted constructed wetland (CW), the low pollutant concentrations and the low COD/N ratios (chemical oxygen demand: total nitrogen in influent), make the biological treatment more difficult. It is expected that root exudates drive microbial-based transformations within plant rhizosphere. In this research, the roles of root exudates of three aquatic plants (Phragmites australis, Typha angustifolia and Cyperus alternifolius) in improving the growth of heterotrophic denitrifying bacteria were determined in a micro-polluted CW. In studied root rhizospheres, the total organic carbon (TOC) released from the plant roots varied significantly among plant species and seasons; the average TOC ranged from 0.1715 to 0.9221mgg−1rootDMd−1, which could fuel a denitrification rate of approximately 156–841kgNO3−-Nha−1year−1 if all were used by the denitrifying bacteria; the abundances of nirK- and nirS-encoding bacteria were significantly influenced by the concentration of sucrose and glucose (0.869≤r≤0.933, p<0.05), and microbial community richness and diversity had response to root exudates. The results revealed that root exudates can act as endogenous carbon sources for heterotrophic denitrifying bacteria and ultimately determine the microbe distribution patterns in micro-polluted CW.

Nitrogen removal and distribution of ammonia-oxidizing and denitrifying genes in an integrated constructed wetland for swine wastewater treatment

This study investigated nitrogen (N) removal process and microbial community distribution in a full-scale integrated constructed wetland (ICW), which was constructed with a bioreactor (R) and three wetland units (W1, W2, and W3) arranged successively along the flow path. For swine wastewater treatment, ICW showed high N removal efficiency with average removal rate of 98% for NH4+-N and 96% for total nitrogen (TN) from August 2012 to July 2014. In ICW sediments, abundance of amoA gene of ammonia-oxidizing archaea (AOA) was 1 order of magnitude greater than ammonia-oxidizing oxidizing bacteria (AOB), and narG gene was 2 orders of magnitude greater than nosZ. The highest amoA gene abundance of AOA and AOB in R were consistent with the most efficient NH4+-N and TN removal compared to W1, W2, and W3. Both W1 and W3 vegetated with Myriophyllum aquaticum had significantly higher AOA abundance than W2 vegetated with Ipomoea aquatica, Zizania latifolia, and Nasturtium officinale (p<0.05). Responding to the increasing NO3−-N concentrations along the flow path, higher narG and nosZ abundance were observed in W2 and W3 than in R and W1. Principal component analysis revealed that there were clear differences in the relative abundance of terminal restriction fragments. These findings demonstrated that environmental factors such as wetland plants and NH4+-N and NO3−-N concentrations had effects on the distri bution of microbial ammonia oxidizers and denitrifiers. Therefore, the responsible development of functional microbial communities could contribute to efficient N removal in the ICW.

Legacy effects of simulated short-term climate change on ammonia oxidisers, denitrifiers, and nitrous oxide emissions in an acid soil.

Although the effect of simulated climate change on nitrous oxide (N2O) emissions and on associated microbial communities has been reported, it is not well understood if these effects are short-lived or long-lasting. Here, we conducted a field study to determine the interactive effects of simulated warmer and drier conditions on nitrifier and denitrifier communities and N2O emissions in an acidic soil and the longevity of the effects. A warmer (+2.3°C) and drier climate (-7.4% soil moisture content) was created with greenhouses. The variation of microbial population abundance and community structure of ammonia-oxidizing archaea (AOA), bacteria (AOB), and denitrifiers (nirK/S, nosZ) were determined using real-time PCR and high-throughput sequencing. The results showed that the simulated warmer and drier conditions under the greenhouse following urea application significantly increased N2O emissions. There was also a moderate legacy effect on the N2O emissions when the greenhouses were removed in the urea treatment, although this effect only lasted a short period of time (about 60days). The simulated climate change conditions changed the composition of AOA with the species affiliated to marine group 1.1a-associated lineage increasing significantly. The abundance of all the functional denitrifier genes decreased significantly under the simulated climate change conditions and the legacy effect, after the removal of greenhouses, significantly increased the abundance of AOB, AOA (mainly the species affiliated to marine group 1.1a-associated lineage), and nirK and nosZ genes in the urea-treated soil. In general, the effect of the simulated climate change was short-lived, with the denitrifier communities being able to return to ambient levels after a period of adaptation to ambient conditions. Therefore, the legacy effect of simulated short-time climate change conditions on the ammonia oxidizer and denitrifier communities and N2O emissions were temporary and once the conditions were removed, the microbial communities were able to adapt to the ambient conditions.

Linkage between N2O emission and functional gene abundance in an intensively managed calcareous fluvo-aquic soil

The linkage between N2O emissions and the abundance of nitrifier and denitrifier genes is unclear in the intensively managed calcareous fluvo-aquic soils of the North China Plain. We investigated the abundance of bacterial amoA for nitrification and narG, nirS, nirK, and nosZ for denitrification by in situ soil sampling to determine how the abundance of these genes changes instantly during N fertilization events and is related to high N2O emission peaks. We also investigated how long-term incorporated straw and/or manure affect(s) the abundance of these genes based on a seven-year field experiment. The overall results demonstrate that the long-term application of urea-based fertilizer and/or manure significantly enhanced the number of bacterial amoA gene copies leading to high N2O emission peaks after N fertilizer applications. These peaks contributed greatly to the annual N2O emissions in the crop rotation. A significant correlation between annual N2O emissions and narG, nirS, and nirK gene numbers indicates that the abundance of these genes is related to N2O emission under conditions for denitrification, thus partly contributing to the annual N2O emissions. These findings will help to draw up appropriate measures for mitigation of N2O emissions in this ‘hotspot’ region.