Denitrification Enzyme Activity Research Articles

Leaf-cutter ants engineer large nitrous oxide hot spots in tropical forests.

Though tropical forest ecosystems are among the largest natural sources of the potent greenhouse gas nitrous oxide (N2O), the spatial distribution of emissions across landscapes is often poorly resolved. Leaf cutter ants (LCA; Atta and Acromyrmex, Myrmicinae) are dominant herbivores throughout Central and South America, and influence multiple aspects of forest structure and function. In particular, their foraging creates spatial heterogeneity by concentrating large quantities of organic matter (including nitrogen, N) from the surrounding canopy into their colonies, and ultimately into colony refuse dumps. Here, we demonstrate that refuse piles created by LCA species Atta colombica in tropical rainforests of Costa Rica provide ideal conditions for extremely high rates of N2O production (high microbial biomass, potential denitrification enzyme activity, N content and anoxia) and may represent an unappreciated source of heterogeneity in tropical forest N2O emissions. Average instantaneous refuse pile N2O fluxes surpassed background emissions by more than three orders of magnitude (in some cases exceeding 80 000 µg N2O-N m-2 h-1) and generating fluxes comparable to or greater than those produced by engineered systems such as wastewater treatment tanks. Refuse-concentrating Atta species are ubiquitous in tropical forests, pastures and production ecosystems, and increase density strongly in response to disturbance. As such, LCA colonies may represent an unrecognized greenhouse gas point source throughout the Neotropics.

Relationships between nitrogen removal processes and functional microorganisms in the rhizosphere soil in a horizontal surface flow constructed wetland

Plant species could significantly affect the nitrogen removal processes mediated by microorganisms in constructed wetlands. However, the links between nitrogen removal processes in the rhizosphere and the related functional microorganisms in a horizontal surface flow constructed wetland in winter remain poorly understood. In this study we collected 24 rhizosphere soils from Typha orientalis and Phragmites australis to evaluate potential nitrogen removal activities, namely the potential nitrification rate (PNR) and denitrification enzyme activity (DEA), and their relationship with functional genes (i.e. nitrate reductase, nirS, and ammonia mono-oxygenase, amoA, of ammonia-oxidising archaea, AOA, and ammonia-oxidising bacteria, AOB) in denitrifiers and nitrifiers in winter. DEA and PNR were significantly higher in the rhizosphere soil of T. orientalis than P. australis, which was due to the higher abundance of nitrifiers and denitrifiers in the rhizosphere of T. orientalis. AOB were the major predictor of PNR in rhizosphere soil of T. orientalis, whereas AOA were more important for P. australis. In addition, denitrifiers containing the nirS gene were found to be the main drivers of DEA, and AOA and AOB also contributed to the denitrification process in the rhizosphere soil of both plants. Furthermore, the abundance of nitrifiers was significantly affected by the C:N ratio, soil organic matter and moisture, whereas the abundance of denitrifiers was affected by soil moisture and pH.

Rapid increases in soil pH solubilise organic matter, dramatically increase denitrification potential and strongly stimulate microorganisms from the Firmicutes phylum.

Rapid and transient changes in pH frequently occur in soil, impacting dissolved organic matter (DOM) and other chemical attributes such as redox and oxygen conditions. Although we have detailed knowledge on microbial adaptation to long-term pH changes, little is known about the response of soil microbial communities to rapid pH change, nor how excess DOM might affect key aspects of microbial N processing. We used potassium hydroxide (KOH) to induce a range of soil pH changes likely to be observed after livestock urine or urea fertilizer application to soil. We also focus on nitrate reductive processes by incubating microcosms under anaerobic conditions for up to 48 h. Soil pH was elevated from 4.7 to 6.7, 8.3 or 8.8, and up to 240-fold higher DOM was mobilized by KOH compared to the controls. This increased microbial metabolism but there was no correlation between DOM concentrations and CO2 respiration nor N-metabolism rates. Microbial communities became dominated by Firmicutes bacteria within 16 h, while few changes were observed in the fungal communities. Changes in N-biogeochemistry were rapid and denitrification enzyme activity (DEA) increased up to 25-fold with the highest rates occurring in microcosms at pH 8.3 that had been incubated for 24-hour prior to measuring DEA. Nitrous oxide reductase was inactive in the pH 4.7 controls but at pH 8.3 the reduction rates exceeded 3,000 ng N2–N g−1 h−1 in the presence of native DOM. Evidence for dissimilatory nitrate reduction to ammonium and/or organic matter mineralisation was observed with ammonium increasing to concentrations up to 10 times the original native soil concentrations while significant concentrations of nitrate were utilised. Pure isolates from the microcosms were dominated by Bacillus spp. and exhibited varying nitrate reductive potential.

Forest canopy restoration has indirect effects on litter decomposition and no effect on denitrification

AbstractForest restoration has potential to recover degraded ecosystem functions in disturbed environments. Decomposition and denitrification are two critical functions involved in forest nutrient cycling that are often compromised in degraded ecosystems. As forest canopy structure develops following initial plantings, it may indirectly impact ecosystem functions by altering abiotic conditions. It is likely, however, that there are other abiotic factors that affect decomposition and denitrification that are unrelated to forest canopy structure. Here, we aimed to determine whether forest canopy openness, topography, and soil sand content would affect litter decomposition and denitrification by regulating the microclimate, the herbaceous plant layer, soil chemistry, and soil moisture. Research occurred in restored native temperate rainforest patches in two New Zealand cities. Urban forests are an excellent context for measuring impact of canopy restoration on ecosystem properties such as microclimate due to the extreme swings in city conditions (e.g., urban heat island). Decomposition rates were determined using leaf litter bags and denitrification rates through denitrification enzyme activity assays. We used structural equation modeling to quantify the direct and indirect drivers of these ecosystem functions. Results indicated that decomposition rates were positively related to soil moisture, relative humidity, and herbaceous plant cover. Interestingly, forest canopy openness indirectly affected decomposition through counteracting forces, meaning greater canopy openness in young forests permitted dense herbaceous plant growth which enhanced decomposition, while less canopy openness in older forests enhanced humidity levels which increased decomposition. Denitrification was negatively related to soil pH and positively related to soil moisture, but these abiotic factors were unrelated to the forest canopy. Discovering drivers of ecosystem functions can improve approaches to the restoration of degraded ecosystems, especially in disturbed urban areas. Identifying counteracting effects on ecosystem functions could improve management by focusing restoration actions on specific drivers to elicit desired changes. Some ecosystem processes, like denitrification, are not affected by forest canopy restoration or management, but are instead driven by edaphic and landscape factors.

Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest.

Acid rain can cause severe effects on soil biota and nutrient biogeochemical cycles in the forest ecosystem, but how plant-symbiotic ectomycorrhizal fungi will modulate the effects remains unknown. Here, we conducted a full factorial field experiment in a Masson pine forest by simultaneously controlling the acidity of the simulated rain (pH 5.6 vs. pH 3.5) and the ectomycorrhizal fungi Pisolithus tinctorius inoculation (non-inoculation vs. inoculation), to investigate the effects on ammonia oxidizers and denitrifiers. After 10 months, compared with the control (rain pH 5.6, and non-inoculation), simulated acid rain (pH 3.5) reduced soil nutrient content, decreased archaeal amoA gene abundance and inhibited denitrification enzyme activity. Also, simulated acid rain altered the community compositions of all the examined functional genes (archaeal amoA, bacterial amoA, nirK, nirS and nosZ). However, inoculation with ectomycorrhizal fungi under acid rain stress recovered soil nutrient content, archaeal amoA gene abundance and denitrification enzyme activity to levels comparable to the control, suggesting that ectomycorrhizal fungi inoculation ameliorates simulated acid rain effects. Taken together, ectomycorrhizal fungi inoculation - potentially through improving soil substrate availability - could alleviate the deleterious effects of acid rain on nitrogen cycling microbes in forest soils.

Effect of Silver Nanoparticles on Denitrification and Functional Gene Abundances of Sediment in Dagu River Estuary and Northwest of Jiaozhou Bay

The influence of silver nanoparticles (AgNPs) on the denitrification performance, enzyme activity, and functional gene relative abundances of sediment was investigated based on the methods of laboratory simulation incubation in the Dagu River estuary and bay area in the northwest of Jiaozhou Bay. The different dosages of AgNPs (i.e., 0, 135, and 1350 mg·L-1 in final concentration) was added to the incubation system containing surface sediments and in-situ bottom water. During six days' incubation, the concentrations of NO3- and NO2-, NO3-, and NO2- reductases activity, and relative abundances of narG and nirS genes were measured to explore the effects of AgNPs on denitrification and its mechanism. The results showed that AgNPs significantly inhibited NO3- and NO2- reductive capacity, NO3- and NO2- reductase activity, and narG and nirS gene relative abundances, which led to aggravated accumulation of NO2-. The inhibition of NO2- reductase was significantly greater than that of NO3- reductase, and the inhibition of the nirS gene was significantly higher than that of the narG gene. The inhibition of NO3- reduction was mainly ascribed to the inhibition of functional genes, but the inhibition of NO2- reduction was mainly due to the inhibition of reductase activity. The inhibition of NO3- and NO2- reductive capacity, NO3- reductase activity, and narG and nirS gene relative abundances in the northwest of Jiaozhou Bay was significantly higher than that in the Dagu River estuary.

Relative importance of landscape versus local wetland characteristics for estimating wetland denitrification potential.

Wetlands can be significant sinks for Nr, via denitrification. There is a lack of understanding about factors controlling denitrification. Research suggests that hydrology, geomorphology, and nitrogen loading are dominant controls. We compared site-scale characteristics with denitrification enzyme activity (DEA) in wetlands along gradients of drainage basin land use to explore the relative importance of landscape and site-scale factors for determining denitrification potential. DEA rates ranged between 0.01-1.69 (μg N gdw-1 hr-1), with most sites falling at the lower end. Sites with higher DEA rates had higher percentages of soil carbon and nitrogen, concentrations of soil extractable NO3 and percent loss on ignition. Sites with upstream agricultural activity had higher DEA rates than more natural sites, but there existed a wide range of DEA rates along both agricultural and urban land gradients. When multiple site and landscape-scale explanatory factors were compared to DEA rates, two site and one landscape scale characteristic (Soil NO3, Soil Percent N, and Percent Agriculture) had significant (p<0.001, cum. r2 = 0.77) correlations with DEA rates. Our results suggest that DEA is controlled mainly by local-scale site characteristics with more work needed to determine the interdependencies and relative importance among these and potentially related landscape-scale factors.

Control of soil N cycle processes by Pteridium aquilinum and Erica cinerea in heathlands along a pH gradient

AbstractNitrate is a limiting resource in heathland acid soils. Nitrate levels increase in heathland soils after Pteridium aquilinum invasions, and this species is assumed to biologically control nitrogen cycle processes, thus increasing nitrate availability. We compared how P. aquilinum (bracken) and Erica cinerea (bell heather) modify processes driving nitrate availability along a soil pH gradient in a Natura 2000 reserve facing bracken invasion. Soil nitrate and ammonium concentrations, substrate‐induced respiration (SIR), denitrification and nitrification enzyme activities (DEA and NEA, respectively), root procyanidin concentrations, and denitrification inhibition by procyanidins were measured on five sites under P. aquilinum and E. cinerea stands. NEA and nitrate levels were higher, and ammonium levels and SIR lower, for P. aquilinum in the most acid soils. Procyanidins from both species induced the same level of denitrification inhibition, soil nitrate being correlated with root procyanidin concentration for both species. Soil nitrate correlated with NEA only for P. aquilinum. Our results show that both species increased procyanidin production in the most acid soils, thereby reducing denitrification and decreasing nitrate loss, this process being more efficient for E. cinerea. However, P. aquilinum additionally increased nitrification, and this double control on nitrification and denitrification was very efficient to increase soil nitrate availability in the most acid soils. This may participate to the success of P. aquilinum invasions in heathlands. This shows that approaches for bracken control in heathlands should better account for belowground processes and, more generally, that biological denitrification inhibition by plants may be a widespread phenomenon influencing soil N dynamics in N‐poor environments.

Effect of the nitrification inhibitor (3, 4-dimethylpyrazole phosphate) on the activities and abundances of ammonia-oxidizers and denitrifiers in a phenanthrene polluted and waterlogged soil

Through a 60-day microcosm incubation, the effect of 3, 4-dimethylpyrazole phosphate (DMPP) on the activities and abundances of ammonia-oxidizers and denitrifiers in phenanthrene-polluted soil was investigated. Five treatments were conducted for clean soil (CK), phenanthrene added (P), phenanthrene and DMPP added (PD), phenanthrene and urea added (PU), and phenanthrene, urea, and DMPP added (PUD) soils. The results indicate that the potential nitrification rate (PNR) in the P treatment was significantly higher than that in the PD treatment only on day 7, whereas the PNR in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. The abundance of soil ammonia-oxidizing bacteria (AOB) in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. Moreover, the abundance of AOB but rather than the ammonia-oxidizing archaea (AOA) had significantly positive correlation with soil PNR (P < 0.05). DMPP showed no obvious effect on the soil denitrification enzyme activity (DEA), which could have inhibited the abundances of denitrification-related narG, nirS, and nirK genes. The results of this study should provide a deeper understanding of the interaction between soil polycyclic aromatic hydrocarbons (PAH) contamination, ammonia oxidization, and denitrification, and offer valuable information for assessing the potential contribution of denitrification for soil PAH elimination.

Sedimentary Environment Influences Ecosystem Response to Nutrient Enrichment

As coastal catchment land use intensifies, estuaries receive increased nutrient and sediment loads, resulting in habitats that are dominated by muddy organic-rich sediments. Increased mud (i.e. silt-clay (particles < 63 μm)) content has been associated with negative effects on soft sediment biodiversity and ecosystem functioning, but the simultaneous impact of nutrient enrichment on ecosystem response is unclear. Nutrient recycling and denitrification in estuarine soft sediments represent important ecosystem functions regenerating nutrients for primary producers and regulating the ability to remove excess terrestrially derived nitrogen. To test the effect of sedimentary environment on ecosystem resilience to nutrient perturbation, we experimentally enriched sediments with slow release fertiliser across an intertidal sedimentary gradient (0–24% mud content). The enrichment successfully elevated pore water ammonium concentrations (median 36 × control) to levels representative of enriched estuaries. Findings show that the sedimentary environment can influence ecosystem function response to nutrient stress. In particular, denitrification enzyme activity was suppressed by nutrient enrichment, but the effect was greater as sediment mud content increased. Furthermore, compared with sandy sediments, sediments with high mud content may restrict nutrient processing (release, uptake or transformation of organic nutrients by the benthos) facilitating ecosystem shifts toward eutrophication. These results show the value of investigating the impacts of stressors in different environmental settings and demonstrate that land use practices that increase the proportion of muddy habitats in estuaries may reduce denitrification which in turn may reduce ecosystem resilience to eutrophication.

Nitrogen-cycling microbial community functional potential and enzyme activities in cultured biofilms with response to inorganic nitrogen availability.

Biofilms mediate crucial biochemical processes in aquatic ecosystems. It was hypothesized that eutrophication may promote the growth of biofilms, resulting in larger numbers of functional genes. However, the metabolic activity and the roles of biofilms in N cycling will be affected by ambient inorganic nitrogen availability, not by the abundance of functional genes. Biofilms were cultured either with replete inorganic nitrogen (N-rep) or without exogenous inorganic nitrogen supply (N-def) in a flow incubator, and the N-cycling gene abundances (nifH, N2 fixation; amoA, ammonia oxidation, archaea and bacteria; nirS and nirK, denitrification) and enzyme activities (nitrogenase and nitrate reductase) were analyzed. The results showed that, comparing the N-def and N-rep biofilms, the former contained lower nifH gene abundance, but higher nitrogenase activity (NA), while the latter contained higher nifH gene abundance, but lower NA. Different patterns of NA diel variations corresponded to the dynamic microbial community composition and different stages of biofilm colonization. Ammonia oxidizing bacteria (AOB), detected only in N-def biofilms, were responsible for nitrification in biofilms. N-rep biofilms contained high nirS and nirK gene abundance and high denitrification enzyme activity, but N-def biofilms contained significantly lower denitrification gene abundance and activity. In general, the strong N2 fixation in N-def biofilms and strong denitrification in N-rep biofilms assured the balance of aquatic ecosystems. The results suggested that evaluation of the functional processes of N cycling should not only focus on genetic potential, but also on the physiological activity of biofilms.

Effect of Nitrate Amendment on Soil Denitrification Activity and Anthracene Anaerobic Degradation

The degradation of soil polycyclic aromatic hydrocarbons (PAHs) under denitrification is one of the most important pathways for anaerobic PAH elimination, but little is known about the effect of nitrate (the terminal electron acceptor for denitrification) on soil denitrification activity and PAH degradation under anaerobic conditions. In this study, the effect of nitrate on soil anthracene anaerobic degradation and denitrification activity was investigated through an anaerobic microcosm experiment. Two groups of treatments without (N0) and with (N30) nitrate (30 mg·kg-1) amendment were conducted. Each group contained three treatments with different anthracene concentrations (0, 15, and 30 mg·kg-1, denoted as A0, A15, and A30, respectively). Therefore, a total of six treatments (N0A0, N0A15, N0A30, N30A0, N30A15, and N30A30) were incubated in darkness at 25℃ for 45 days, and the production rates of N2O and CO2, abundances of denitrification related genes (narG:periplasmic nitrate reductase gene; nirK:copper-containing nitrite reductase gene; and nirS:cd1-nitrite reductase gene), and soil anthracene content were measured at 3, 7, 14, and 45 days. The results indicated that the intensive denitrification enzyme activity in each treatment was only detected at day 3, which could be significantly enhanced by both nitrate and anthracene amendments. Subsequently, a sharp decline of denitrification enzyme activity was observed in each treatment, while anthracene showed an obvious inhibition of soil denitrification enzyme activity. The result of a two-way ANOVA also indicated that nitrate, anthracene, and their interactions had significant effects on soil denitrification enzyme activity. The result of a quantitative-PCR indicated that, during the incubation, the abundances of narG and nirS exhibited an increasing tendency, but the abundance of nirK was relatively constant compared with its former counterparts. The final removal rate of anthracene under anaerobic soil environment was in the range of 33.83%-55.01%, and neither the final removal rate nor the degradation rate of anthracene could be significantly affected by nitrate amendment during incubation. The anthracene degradation rates in the higher anthracene containing treatments (N0A30 and N30A30) were significantly higher than those in the lower anthracene containing treatments (N0A15 and N30A15). In summary, nitrate amendments had no effect on soil anthracene anaerobic degradation but could significantly affect soil denitrification enzyme activity and the abundance of denitrification related narG and nirS genes.

An increased ratio of fungi to bacteria indicates greater potential for N2O production in a grazed grassland exposed to elevated CO2

Nitrous oxide (N2O) is an important greenhouse gas and emissions of N2O have been shown to increase under elevated CO2 (eCO2) resulting in a positive feedback on climate change. CO2-driven increases under grassland have often been associated with greater N2O emitted during denitrification. We examined the soils from a Free Air Carbon Dioxide Enrichment (FACE) experiment on grassland on the west coast of the North Island of New Zealand that had received long-term exposure to elevated CO2. Importantly, the grassland was grazed thus representing much of the world’s grassland situation and providing data for a land use that has not been well studied. We conducted soil incubations where a fungicide and bactericide were used to isolate the contribution of bacteria and fungi to potential N2O production using denitrification enzyme activity (DEA). We found greater gene abundance of fungi under eCO2 and reduced bacterial gene abundance. N2O DEA was dominated by fungi in both ambient and elevated CO2. Total potential N2O emissions were 49% higher under eCO2 entirely due to greater emissions from the fungal component. An increasing fungal contribution to N2O emissions presents a challenge to mitigation as, to date, mitigations have largely been targeted at bacteria.

Greenhouse gas emissions from a Cu-contaminated soil remediated by in situ stabilization and phytomanaged by a mixed stand of poplar, willows, and false indigo-bush

ABSTRACTPhytomanagement of trace element-contaminated soils can reduce soil toxicity and restore soil ecological functions, including the soil gas exchange with the atmosphere. We studied the emission rate of the greenhouse gases (GHGs) CO2, CH4, and N2O; the potential CH4 oxidation; denitrification enzyme activity (DEA), and glucose mineralization of a Cu-contaminated soil amended with dolomitic limestone and compost, alone or in combination, after a 2-year phytomanagement with a mixed stand of Populus nigra, Salix viminalis, S. caprea, and Amorpha fruticosa. Soil microbial biomass and microbial community composition after analysis of the phospholipid fatty acids (PLFA) profile were determined. Phytomanagement significantly reduced Cu availability and soil toxicity, increased soil microbial biomass and glucose mineralization capacity, changed the composition of soil microbial communities, and increased the CO2 and N2O emission rates and DEA. Despite such increases, microbial communities were evolving toward less GHG emission per unit of microbial biomass than in untreated soils. Overall, the aided phytostabilization option would allow methanotrophic populations to establish in the remediated soils due to decreased soil toxicity and increased nutrient availability.

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.

Denitrification along the Stream-Riparian Continuum in Restored and Unrestored Agricultural Streams.

Although floodplain restoration may improve stream-riparian connectivity and increase channel stability through cross-vane installation, stream bank regrading, and buffer revegetation, the impact of these geomorphic alterations on denitrification across the aquatic-terrestrial interface is poorly understood. Seasonal denitrification enzyme activity (DEA) was compared in stream-riparian ecotones of four streams with varying hydrologic connection, riparian vegetation composition, and agricultural influence. Riparian and stream DEA was generally higher in sites adjacent to agricultural fields due to proximity to nitrate source. Mean DEA was higher in the dormant season (riparian: 928 ± 116 ng N g dry mass [DM] h, stream: 108 ± 149 ng N g DM h) than in the growing season (riparian: 355 ± 55 ng N g DM h, stream: 45 ± 40 ng N g DM h) and was influenced by antecedent precipitation, soil texture, and landscape characteristics (land use, vegetation type, topography) in multivariate models. Hot spots of denitrification occurred at the confluence of fine soil textures with high organic matter and moisture, often at low-lying areas of the landscape. At the restored site, we observed enhanced denitrification in the reconnected floodplain but decreased stream denitrification capacity in pools. Across all sites, streams had lower DEA than riparian zones, stressing the importance of restoration approaches designed to conserve riparian areas and maintain hydrologic connectivity. Easily obtainable data including precipitation, adjacent land use, vegetation, and stream and floodplain geomorphology reasonably predicted denitrification potential compared with more complex models, showing potential for application in water resources and nitrogen management.

Denitrifying bacterial communities display different temporal fluctuation patterns across Dutch agricultural soils.

Considering the great agronomic and environmental importance of denitrification, the aim of the present study was to study the temporal and spatial factors controlling the abundance and activity of denitrifying bacterial communities in a range of eight agricultural soils over 2years. Abundance was quantified by qPCR of the nirS, nirK and nosZ genes, and the potential denitrification enzyme activity (DEA) was estimated. Our data showed a significant temporal variation considerably high for the abundance of nirK-harboring communities, followed by nosZ and nirS communities. Regarding soil parameters, the abundances of nosZ, nirS and nirK were mostly influenced by organic material, pH, and slightly by NO3-, respectively. Soil texture was the most important factor regulating DEA, which could not be explained by the abundance of denitrifiers. Analyses of general patterns across lands to understand the soil functioning is not an easy task because the multiple factors influencing processes such as denitrification can skew the data. Careful analysis of atypical sites are necessary to classify the soils according to trait similarity and in this way reach a better predictability of the denitrifiers dynamics.

Base cations, K+ and Ca2+, have contrasting effects on soil carbon, nitrogen and denitrification dynamics as pH rises

Full denitirifcation of NO3− to N2 is most likely when soil pH is elevated and oxygen levels are low. In the period immediately following urine deposition, soil pH and dissolved organic carbon (DOC) concentrations rapidly increase, coupled with O2 consumption - conditions conducive to denitrification and gaseous N-loss - yet the potential for rapid denitrification during this early period post urine deposition has received little attention. In addition, recent research has shown that the amount of DOC (and N) released as pH increases is affected by the valence of the accompanying base cation, with monovalent cations releasing more. Liming has been used to positively influence N2O/(N2O + N2) ratios in the later stages of N-transformations post urine deposition, yet the relative amounts of DOC (and N) released is likely to be affected in the presence of divalent Ca2+ potentially lowering N-transformation potential.Soil pH in anaerobic microcosms was adjusted to values between 4.5 and 9.5 using either Ca(OH)2 as a liming agent, or KOH as an analog of urine derived NH4OH. After 16 h incubation, up to ∼6800 μg g−1 of DOC and ∼480 μg g−1 of dissolved organic nitrogen (DON) was solubilized by KOH amendment which was ∼10-fold higher than Ca(OH)2 ammendment, while NH4+ and NO3− increased to concentrations well above native soil concentrations. In Ca(OH)2 amended microcosms, denitrification enzyme activity (DEA) assays produced a maximum of 875 ng N2O-N g−1 h−1 at pH 7.2 with respiration rates <10 μg CO2-C g−1 h−1. In contrast, DEA and respiration continually climbed in KOH amended microcosms with rates up to 4798 ng N2O-N g−1 h−1 and 47 μg CO2-C g−1 h−1 at pH 8.7 and 8.9, respectively.By using KOH as proxy for NH4OH derived from urea hydrolysis, this research has shown that rapid denitrification could occur shortly after urine deposition along with substantial DOC and DON solubilisation and N-mineralisation. However, elevated soil pH combined with excess Ca2+ from liming might actually suppress denitrification potential. We propose that excess Ca2+ affects how soil microbes access and process DOC and DON at elevated pH, with Ca2+ acting to stabilize DOC and DON. Our results emphasize that although liming promotes soil conditions conducive to denitrification, DEA can be markedly affected by base type and prevailing soil chemical conditions.

Nitrate removal, spatiotemporal communities of denitrifiers and the importance of their genetic potential for denitrification in novel denitrifying bioreactors

Nitrate treatment performance and denitrification activity were compared between denitrifying biological filters (DNBFs) based on dewatered alum sludge (DAS) and neutralized used acid (NUA). The spatiotemporal distribution of denitrifying genes and the genetic potential associated with denitrification activity and nitrate removal in both DNBFs were also evaluated. The removal efficiency of NUA–DNBF increased by 8% compared with that of DAS–DNBF, and the former NUA–DNBF emitted higher amount of N2O. Analysis of abundance and composition profiles showed that denitrifying gene patterns varied more or less in two matrices with different depths at three sampling times. Burkholderiales, Rhodocyclales, and Rhizobiales were the most commonly detected in both media during stable periods. Denitrification was determined by the abundance of specific genes or their ratios as revealed by controlling factors. The enhanced nitrate removal could be due to increasing qnosZ or decreasing ∑qnir/qnosZ. Furthermore, NUA–DNBF solely reduced nitrate by increasing the denitrification enzyme activity.

Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil

Nitrifiers and denitrifiers are the key drivers of N cycling in paddy soil. Little is known about the effects of different fertilization methods, especially side bar fertilization, on the community of nitrifiers and denitrifiers in paddy soils. Here we assess the relationships between soil physicochemical properties, denitrification and nitrification activities, and the underlying microbial communities in a surface layer of paddy field soil treated with different fertilization methods. Soil was unfertilized (control), treated with conventional chemical fertilizer (CF), CF plus pig manure (MC), or slow-release fertilizer (SR), or by slow-release side bar fertilization (SB). Soil was sampled after one season of early and late rice growth. We determined soil physicochemical properties, potential nitrification rates (PNR), and denitrification enzyme activities (DEA). Ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities were assessed via their ammonia monooxygenase (amoA) genes, and denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ). Quantitative PCR was used to assess gene abundance, terminal restriction fragment polymorphism (T-RFLP) to investigate fertilization effects on microbial communities, and clone library sequencing and phylogenetic analysis to explore the taxonomic diversity of the nitrifiers and denitrifiers. Fertilization significantly increased the amount of NH4 +-N in the soil of SB and MC treatments, whereas MC lowered the NO3 −-N level. DEA was higher for MC and CF than the other treatments. The PNR in MC-treated soil was significantly lower than that in CF-treated soil. There were no significant differences in AOA and nirS copy numbers; however, nirK and nosZ copy numbers were higher for MC compared with CF. The number of AOA terminal restriction fragments (TRFs) increased significantly with N addition and reached the highest level for SB, whereas the number of AOB TRFs did not change significantly between treatments. Similarly, the number of nirK TRFs increased under fertilization, with the highest number obtained for SR; however, no significant change was observed for nirS and nosZ TRFs across different treatments, except for their relative abundance. All AOA amoA genotypes were in archaeal group 1.1b, whereas 95% AOB were in Nitrosospira cluster 3a. More than 40% of nirS OTUs were affiliated with Herbaspirillum, a key N-cycle player in this paddy soil. The SB and MC treatments had significant effects on soil N, DEA, and PNR levels, and affected the community of N-functional microbes. SB in combination with pig manure would contribute to the improvement of paddy soil fertility.