Heterotrophic and autotrophic nitrification in two acid pasture soils

Laboratory incubation experiments, using ¹⁵N-labeling techniques and simple analytical models, were conducted to measure heterotrophic and autotrophic nitrification rates in two acid soils (pH 4.8-5.3; 1/5 in H₂O) with high organic carbon contents (6.2-6.8% in top 5 cm soil). The soils were from pastures located near Maindample and Ruffy in the Northeast Victoria, Australia. Gross rates of N mineralization, nitrification and immobilization were measured. The gross rates of autotrophic nitrification were 0.157 and 0.119 μg N g⁻¹ h⁻¹ and heterotrophic nitrification rates were 0.036 and 0.009 μg N g⁻¹ h⁻¹ for the Maindample and Ruffy soils, respectively. Heterotrophic nitrification accounted for 19% and 7% of the total nitrification in the Maindample and Ruffy soils, respectively. The heterotrophic nitrifiers used organic N compounds and no as the substrate for nitrification.     

Effects of 3,4-dimethylpyrazole phosphate (DMPP) on the abundance of ammonia oxidizers and denitrifiers in two different intensive vegetable cultivation soils

Purpose

Nitrification and denitrification in the N cycle are affected by various ammonia oxidizers and denitrifying microbes in intensive vegetable cultivation soils, but our current understanding of the effect these microbes have on N₂O emissions is limited. The nitrification inhibitor, 3,4-dimethylpyrazole phosphate (DMPP), acts by slowing nitrification and is used to improve fertilizer use efficiency and reduce N losses from agricultural systems; however, its effects on nitrifier and denitrifier activities in intensive vegetable cultivation soils are unknown.

Materials and methods

In this study, we measured the impacts of DMPP on N₂O emissions, ammonia oxidizers, and denitrifying microbes in two intensive vegetable cultivation soils: one that had been cultivated for a short term (1 year) and one that had been cultivated over a longer term (29 years). The quantitative PCR technique was used in this study. Three treatments, including control (no fertilizer), urea alone, and urea with DMPP, were included for each soil. The application rates of urea and DMPP were 1800 kg ha^?1 and 0.5% of the urea-N application rate.

Results and discussion

The application of N significantly increased N₂O emissions in both soils. The abundance of ammonia-oxidizing bacteria (AOB) increased significantly with high rate of N fertilizer application in both soils. Conversely, there was no change in the growth rate of ammonia-oxidizing archaea (AOA) in response to the applied urea despite the presence of larger numbers of AOA in these soils. This suggests AOB may play a greater role than AOA in the nitrification process, and N₂O emission in intensive vegetable cultivation soils. The application of DMPP significantly reduced soil NO₃^?-N content and N₂O emission, and delayed ammonia oxidation. It greatly reduced AOB abundance, but not AOA abundance. Moreover, the presence of DMPP was correlated with a significant decrease in the abundance of nitrite reductase (nirS and nirK) genes.

Conclusions

Long-term intensive vegetable cultivation with heavy N fertilization altered AOB and nirS abundance. In vegetable cultivation soils with high N levels, DMPP can be effective in mitigating N₂O emissions by directly inhibiting both ammonia oxidizing and denitrifying microbes.

     

Effects of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on abundance and activity of ammonia oxidizers in soil

Biology and Fertility of Soils - Recent evidence from several environments suggest that besides autotrophic ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) are also able to...     

Effect of urease and nitrification inhibitors on N transformation, gaseous emissions of ammonia and nitrous oxide, pasture yield and N uptake in grazed pasture system

Nitrogen (N) losses via nitrate (NO₃⁻) leaching, ammonia (NH₃) volatilization and nitrous oxide (N₂O) emissions from grazed pastures in New Zealand are one of the major contributors to environmental degradation. The use of N inhibitors (urease and nitrification inhibitors) may have a role in mitigating these N losses. A one-year field experiment was conducted on a permanent dairy-grazed pasture site at Massey University, Palmerston North, New Zealand to quantify these N losses and to assess the effect of N inhibitors in reducing such losses during May 2005-2006. Cow urine at 600 kg N ha⁻¹ rate with or without urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) or (trade name “Agrotain”) (3 L ha⁻¹), nitrification inhibitor dicyandiamide (DCD) (7 kg ha⁻¹) and the use of double inhibitor (DI) containing a combination of both Agrotain and DCD (3:7) were applied to field plots in autumn, spring and summer. Pasture production, NH₃ and N₂O fluxes, soil mineral N concentrations, microbial biomass C and N, and soil pH were measured following the application of treatments during each season. All measured parameters, except soil microbial biomass C and N, were influenced by the added inhibitors during the three seasons. Agrotain reduced NH₃ emissions over urine alone by 29%, 93% and 31% in autumn, spring and summer respectively but had little effect on N₂O emission. DCD reduced N₂O emission over urine alone by 52%, 39% and 16% in autumn, spring and summer respectively but increased NH₃ emission by 56%, 9% and 17% over urine alone during those three seasons. The double inhibitor reduced NH₃ by 14%, 78% and 9% and N₂O emissions by 37%, 67% and 28% over urine alone in autumn, spring and summer respectively. The double inhibitor also increased pasture dry matter by 10%, 11% and 8% and N uptake by the 17%, 28% and 10% over urine alone during autumn, spring and summer respectively. Changes in soil mineral N and pH suggested a delay in urine-N hydrolysis with Agrotain, and reduced nitrification with DCD. The combination of Agrotain and DCD was more effective in reducing both NH₃ and N₂O emissions, improving pasture production, controlling urea hydrolysis and retaining N in NH₄⁺ form. These results suggest that the combination of both urease and nitrification inhibitors may have the most potential to reduce N losses if losses are associated with urine and improve pasture production in intensively grazed systems.     

Restricted nitrous oxide emissions by ammonia oxidizers in two agricultural soils following excessive urea fertilization

Journal of Soils and Sediments - Nitrogen (N) fertilizer placement in bands is a widely accepted agricultural practice to increase N use efficiency. An excessive ammonium concentration in a...     

Effects of form of effluent,season and urease inhibitor on ammonia volatilization from dairy farm effluent applied to pasture

Purpose

With land application of farm effluents from cows during housing or milking as an accepted practice, there are increasing concerns over its effect on nitrogen (N) loss through ammonia (NH₃) volatilization. Understanding the relative extent and seasonal variation of NH₃ volatilization from dairy effluent is important for the development of management practices for reducing NH₃ losses. The objectives of this study were to determine potential NH₃ losses from application of different types of dairy effluent (including both liquid farm dairy effluent (FDE) and semi-solid dairy farm manure) to a pasture soil during several contrasting seasons and to evaluate the potential of the urease inhibitor (UI)—N-(n-butyl) thiophosphoric triamide (NBTPT, commercially named Agrotain^®) to reduce gaseous NH₃ losses.

Material and methods

Field plot trials were conducted in New Zealand on an established grazed pasture consisting of a mixed perennial ryegrass (Lolium perenne L.)/white clover (Trifolium repens L.) sward. An enclosure method, with continuous air flow, was used to compare the effects of treatments on potential NH₃ volatilization losses from plots on a free-draining volcanic parent material soil which received either 0 (control) or 100 kg N ha^?1 as FDE or manure (about 2 and 15 % of dry matter (DM) contents in FDE or manure, respectively) with or without NBTPT (0.25 g NBTPT kg^?1 effluent N). The experiment was conducted in the spring of 2012 and summer and autumn of 2013.

Results and discussion

Results showed that application of manure and FDE, both in fresh and stored forms, potentially led to NH₃ volatilization, ranging from 0.6 to 19 % of applied N. Difference in NH₃ losses depended on the season and effluent type. Higher NH₃ volatilization was observed from both fresh and stored manure, compared to fresh and stored FDE. The difference was mainly due to solid contents. The losses of NH₃ were closely related to NH₄ ⁺-N content in the two types of manure. However, there was no relationship between NH₃ losses and NH₄ ⁺-N content in either type of FDE. There was no consistent seasonal pattern, although lower NH₃ losses from fresh FDE and stored FDE applied in spring compared to summer were observed. Potential NH₃ losses from application of fresh FDE or manure were significantly (P?<?0.05) reduced by 27 to 58 % when NBTPT was added, but the UI did not significantly reduce potential NH₃ volatilization from stored FDE or manure.

Conclusions

This study demonstrated that NH₃ losses from application of FDE were lower than from manure and that UIs can be effective in mitigating NH₃ emissions from land application of fresh FDE and manure. Additionally, reducing the application of FDE in summer can also potentially reduce NH₃ volatilization from pasture soil.     

Humic acids buffer the effects of urea on soil ammonia oxidizers and potential nitrification

Humic acids (HAs) play an important role in the global nitrogen cycle by influencing the distribution, bioavailability, and ultimate fate of organic nitrogen. Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in ecosystems and is limited, in part, by the availability of NH₄⁺. We evaluated the impact of HAs on soil AOB in microcosms by applying urea (1.0%, equal to 10 mg urea/g soil) with 0.1% bHA (biodegraded lignite humic acids, equal to 1 mg/g soil), 0.1% cHA (crude lignite humic acids) or no amendment. AOB population size, ammonium and nitrate concentrations were monitored for 12 weeks after urea and HA application. AOB densities (quantified by real-time PCR targeting the amoA) in the Urea treatments increased about ten-fold (the final abundance: 5.02 × 10⁷ copies (g of dry soil)⁻¹) after one week of incubation and decreased to the initial density after 12 weeks incubation; the population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) decreased from 1.12 × 10¹⁰ to 2.59 × 10⁹ copies (g of dry soil)⁻¹ at week one and fluctuated back to the initial copy number at week 12. In the Urea + bHA and Urea + cHA treatments, the AOB densities were 4 and 6 times higher, respectively, than the initial density of approximately 5.07 × 10⁶ copies (g of dry soil)⁻¹ at week 1 and did not change much up to week 4; the total bacteria density changed little over time. The AOB and total bacteria density of the controls changed little during the 12 weeks of incubation. The microbial community composition of the Urea treatment, based on T-RFLP using CCA (canonical correspondence analysis) and pCCA (partial CCA) analysis, was clearly different from those of other treatments, and suggested that lignite HAs buffered the change in diversity and quantity of total bacteria caused by the application of urea to the soil. We hypothesize that HAs can inhibit the change in microbial community composition and numbers, as well as AOB population size by reducing the hydrolysis rate from urea to ammonium in soils amended with urea.     

Effects of the nitrification inhibitor dicyandiamide on soil mineral N, pasture yield, nutrient uptake and pasture quality in a grazed pasture system

Recent lysimeter studies have demonstrated that the nitrification inhibitor, dicyandiamide (DCD), can reduce nitrate (NO) leaching losses from cow urine patches in grazed pasture systems. The objective of this study was to quantify the effects of fine particle suspension (FPS) DCD on soil mineral N components, pasture yield, nutrient uptake and pasture quality under grazed pasture conditions. A field study was conducted on the Lincoln University dairy farm, Canterbury, New Zealand, from 2002 to 2006. FPS DCD was applied to grazed pasture plots at 10 kg ha^?1 in early May in addition to applied cow urine patches at a nitrogen (N) loading rate of 1000 kg N ha^?1, with DCD reapplied in early August. Soil mineral N levels in the urine patches were monitored. Pasture yield, N and cation concentrations and uptake were measured in treatment urine patches and inter‐urine areas of the pasture. Comparisons were made with control plots which did not receive DCD. NO levels under the DCD‐treated urine patches (0–7.5 cm) were in the order of 10 kg N ha^?1 compared with 40–80 kg N ha^?1 under untreated patches, and soil ammonium (NH) levels were consistently higher under the DCD‐treated patches. The DCD significantly and consistently increased pasture yield in both the urine patches, and inter‐urine areas of the pasture in all 4 years of the trial. Mean annual dry matter (DM) yields over 4 years were inter‐urine areas, 10.3; inter‐urine + DCD, 12.4; urine, 12.4 and urine +DCD 16.0 t DM ha^?1, representing an average DM yield increase of 20 and 29% in inter‐urine and urine patch areas, respectively. On a whole paddock basis, the increase in annual DM yield resulting from DCD application was estimated to be 21%. N, calcium (Ca), magnesium (Mg) and potassium (K) concentrations in pasture were unaffected by treatment with DCD; however, total annual uptake of these nutrients by pasture was significantly higher in all years where DCD had been applied. Pasture DM, protein, carbohydrate, metabolizable energy and fibre levels and sward clover content were not affected by treatment with DCD. The results demonstrate the agronomic value of the DCD treatment in addition to the environmental benefits in a grazed pasture system.     

Responses of ureolytic and nitrifying microbes to urease and nitrification inhibitors in selected agricultural soils in Victoria,Australia

Journal of Soils and Sediments - Urease inhibitors (UIs) such as N-(n-butyl)thiophosphoric triamide (NBPT) and nitrification inhibitors (NIs) such as 3,4-dimethylpyrazole phosphate (DMPP) have been...     

The short-term effects of nitrification inhibitors on the abundance and expression of ammonia and nitrite oxidizers in a long-term field experiment comparing land management

Microcosms were set up to evaluate the effect of nitrification inhibitors (DCD, c-PTiO, and NaClO₃) on the abundance and expression of ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as the nitrite-oxidizing bacteria (NOB) Nitrospira and Nitrobacter. Both DCD and NaClO₃ inhibited the net nitrification rate, while c-PTiO had no significant effects, and NaClO₃ had a much greater inhibitory effect (> 60%) in all soils than DCD. No significant changes in total microbial abundance were observed with DCD and NaClO₃. DCD limited only the growth of AOB; however, NaClO₃ inhibited growth of both AOA and Nitrospira-NOB with no significant effects on AOB and Nitrobacter-NOB. Probably NaClO₃ inhibited both ammonia oxidation and nitrite oxidation. This is the first report to reveal the inhibitory effects of NaClO₃ on a specific nitrification process, helping to clarify the ecological niche of nitrifiers and the potential of nitrification inhibitors applied to soil.     

Potential contribution of lysed bacterial cells to phosphorus solubilisation in two rewetted Australian pasture soils

Soil drying renders considerable amounts of phosphorus soluble upon rewetting, which may be partly derived from lysed microbial cells. Using direct bacterial cell counting in water and tetra-sodium pyrophosphate extracts of two Australian pasture soils, we found that almost all extractable cells were lysed following the rewetting of dry soils. The amounts of phosphorus in the lysed cells corresponded closely to the increases in water-extractable phosphorus following soil drying, suggesting that bacterial cell lysis is a major source of the released phosphorus.     

Effect of 7-year application of a nitrification inhibitor, dicyandiamide (DCD), on soil microbial biomass, protease and deaminase activities, and the abundance of bacteria and archaea in pasture soils

Purpose

The nitrification inhibitor dicyandiamide (DCD) has been shown to be highly effective in reducing nitrate (NO₃ ^?) leaching and nitrous oxide (N₂O) emissions when used to treat grazed pasture soils. However, there have been few studies on the possible effects of long-term DCD use on other soil enzyme activities or the abundance of the general soil microbial communities. The objective of this study was to determine possible effects of long-term DCD use on key soil enzyme activities involved in the nitrogen (N) cycle and the abundance of bacteria and archaea in grazed pasture soils.

Materials and methods

Three field sites used for this study had been treated with DCD for 7 years in field plot experiments. The three pasture soils from three different regions across New Zealand were Pukemutu silt loam in Southland in the southern South Island, Horotiu silt loam in the Waikato in the central North Island and Templeton silt loam in Canterbury in the central South Island. Control and DCD-treated plots were sampled to analyse soil pH, microbial biomass C and N, protease and deaminase activity, and the abundance of bacteria and archaea.

Results and discussion

The three soils varied significantly in the microbial biomass C (858 to 542 μg C g^?1 soil) and biomass N (63 to 28 μg N g^?1), protease (361 to 694 μg tyrosine g^?1 soil h^?1) and deaminase (4.3 to 5.6 μg NH₄ ⁺ g^?1 soil h^?1) activity, and bacteria (bacterial 16S rRNA gene copy number: 1.64?×?10⁹ to 2.77?×?10⁹ g^?1 soil) and archaea (archaeal 16S rRNA gene copy number: 2.67?×?10⁷ to 3.01?×?10⁸ g^?1 soil) abundance. However, 7 years of DCD use did not significantly affect these microbial population abundance and enzymatic activities. Soil pH values were also not significantly affected by the long-term DCD use.

Conclusions

These results support the hypothesis that DCD is a specific enzyme inhibitor for ammonia oxidation and does not affect other non-target microbial and enzyme activities. The DCD nitrification inhibitor technology, therefore, appears to be an effective mitigation technology for nitrate leaching and nitrous oxide emissions in grazed pasture soils with no adverse impacts on the abundance of bacteria and archaea and key enzyme activities.     

Effects of acetylene on nitrification and denitrification in two soils during incubation with ammonium nitrate

A loam from the Frilsham and one from the Wickham Series were incubated at 50 and 90 per cent of their water contents at saturation with 100 μg NH₄NO₃-N_g^?1 soil in the presence and absence of C₂H₂ (0.5 per cent, v/v). Acetylene inhibited nitrification in both soils, but had no effect on mineralization of N. No denitrification (measured as the production of N₂O in the presence of C₂H₂) occurred during incubation at 50 per cent saturation. At 90 per cent saturation, denitrification resulted in a loss of 28.4 and 36.7 μg N_g^?1 after 48 h from the Frilsham and Wickham soils, respectively. The concurrent inhibition of nitrification had no effect on the extent of denitrification at this time. In the Wickham soil, NO₃^? was exhausted after 168 h incubation in the presence of C₂H₂ and denitrification was underestimated by 13 μg N_g^?. The data suggested that concurrent inhibition of nitrification during measurement of denitrification using the C₂H₂ inhibition technique is most likely to affect the estimate of denitrification loss when NO₃^?supply is limited by the inhibition of nitrification.     

Production and persistence of urease activity in soils

Addition of urea to Iowa soils did not induce urease activity, but production of urease activity was observed on addition of glucose and other organic materials that promote microbial activity. The persistence of the urease activity produced on addition of these materials varied with the soil, but, with each soil studied, the urease activity after addition of organic materials eventually was identical to that of the unamended soil. No increase or decrease in urease activity was observed when unamended or urea-treated soils were incubated under aerobic conditions for several months. It is concluded that soil constituents protect urease against microbial degradation and other processes leading to inactivation of enzymes and that every soil has a stable level of urease activity determined by the ability of its constituents to provide this protection.     

Contributions of ammonia-oxidizing archaea and bacteria to nitrification in Oregon forest soils

Ammonia oxidation, the first step of nitrification, is mediated by both ammonia-oxidizing archaea (AOA) and bacteria (AOB); however, the relative contributions of AOA and AOB to soil nitrification are not well understood. In this study we used 1-octyne to discriminate between AOA- and AOB-supported nitrification determined both in soil-water slurries and in unsaturated whole soil at field moisture. Soils were collected from stands of red alder (Alnus rubra Bong.) and Douglas-fir (Pseudotsuga menziesii Mirb. Franco) at three sites (Cascade Head, the H.J. Andrews, and McDonald Forest) on acidic soils (pH 3.9–5.7) in Oregon, USA. The abundances of AOA and AOB were measured using quantitative PCR by targeting the amoA gene, which encodes subunit A of ammonia monooxygenase. Total and AOA-specific (octyne-resistant) nitrification activities in soil slurries were significantly higher at Cascade Head (the most acidic soils, pH < 5) than at either the H.J. Andrews or McDonald Forest, and greater in red alder compared with Douglas-fir soils. The fraction of octyne-resistant nitrification varied among sites (21–74%) and was highest at Cascade Head than at the other two locations. Net nitrification rates of whole soil without NH₄⁺ amendment ranged from 0.4 to 3.3 mg N kg⁻¹ soil d⁻¹. Overall, net nitrification rates of whole soil were stimulated 2- to 8-fold by addition of 140 mg NH₄⁺-N kg⁻¹ soil; this was significant for red alder at Cascade Head and the H.J. Andrews. Red alder at Cascade Head was unique in that the majority of NH₄⁺-stimulated nitrifying activity was octyne-resistant (73%). At all other sites, NH₄⁺-stimulated nitrification was octyne-sensitive (68–90%). The octyne-sensitive activity—presumably AOB—was affected more by soil pH whereas the octyne-resistant (AOA) activity was more strongly related to N availability.     

Response of tomato to rates and methods of placement of urea and ammonium sulfate as affected by dwell,a nitrification inhibitor

Abstract

The effectiveness of Dwell, a nitrification inhibitor, was tested in a greenhouse with two fertilizers and three Arizona soils. Tomato plants (Lycopersicum esculentum Mill., cv. Row Pak) were grown in ABS plastic columns 10 cm diameter and 50 cm long containing 4500 g soil. Urea and ammonium sulfate (AS) were applied at 112 and 224 kg N/ha and Dwell (5‐ethoxy‐3‐trichloromethyl‐l,2,4‐thiadiazole) was applied at rates of 0 and 1.27 kg/ha of active ingredient. Both fertilizers and Dwell were added by mixing uniformly or in a band. Dwell increased ? uptake by 41%. The highest ? uptake was with Dwell with the highest urea rate, but Dwell was effective with both fertilizers in all soils. The lowest response to Dwell was with AS‐Laveen loam treatment, whereas the highest response, but the lowest ? uptake, was with urea ‐ Vinton loamy sand treatment. Dwell did not affect the inorganic‐N contents of the soils.     

Preliminary study on the distribution of ammonia oxidizers and their contribution to potential ammonia oxidation in the plant-bed/ditch system of a constructed wetland

Purpose

Ammonia oxidation—as the rate-limiting step of nitrification—has been found to be performed by both ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, how ammonium content and oxidation–reduction status regulate the distribution of ammonia oxidizers in constructed wetlands and their contribution to potential ammonia oxidation rate are still in dispute. This study aimed to explore the effects of ammonium content and oxidation–reduction status on the abundances of AOA/AOB and examine the contributions of AOA and AOB populations to ammonia oxidation rates in the plant-bed/ditch system of a constructed wetland.

Materials and methods

Sampling was carried out in the plant-bed/ditch system of the Shijiuyang Constructed Wetland, China. Three plant-bed soil cores were collected using a soil auger and sampled at depths of 0, 20, and 50 cm in 5-cm increments. Five ditch surface sediments (0–5 cm) were collected along the water flow direction. The abundances of AOA and AOB were investigated by quantitative polymerase chain reaction based on amoA genes. The potential ammonia oxidation rate was determined using the chlorate inhibition method.

Results and discussion

The results showed that AOA outnumbered AOB in the plant-bed surface soil which had lower ammonium content (4.67–7.63 mg kg^?1), but that AOB outnumbered AOA in the ditch surface sediment which had higher ammonium content (14.0–22.9 mg kg^?1). Ammonium content was found to be the crucial factor influencing the relative abundances of AOA and AOB in the surface samples of the plant-bed/ditch system. In the deep layers of the plant bed, AOA abundance outnumbered AOB, though much lower oxidation–reduction potential occurred along the water flow direction. Thus, the oxidation–reduction potential may be another factor influencing the distributions of AOA and AOB in the deep layers of the plant bed without significant difference in ammonium content (p?<?0.05). Moreover, the potential ammonia oxidation rate was significantly dominated by AOB rather than AOA in the plant-bed/ditch system.

Conclusions

The high ammonium content in the ditch sediment likely favored AOB. AOA seemed to persist more readily even under low oxidation–reduction potential in the deep layers of the plant bed. Ammonium content and the oxidation–reduction potential were important parameters influencing the distribution of AOA and AOB in the plant-bed/ditch system of Shijiuyang Constructed Wetland. AOB contributed more to ammonia oxidation than AOA, both in the plant-bed soils (r?=?0.592, p?=?0.0096) and in the ditch sediments (r?=?0.873, p?=?0.0002).