The agricultural sector uses fertilizers such as urea to add more nutrients to the soil needed for plant growth. Although it is cost-effective in crop production, indiscriminate use of nitrate-based fertilizer may result in behavioural, morphological, and physiological alterations on non-target organisms. This study determined the angiogenesis activity in the chorioallantoic membrane of urea-exposed duck embryos. It also investigated the weight, morphometries, and liver histopathology to gather more information on urea fertilizer's toxicity. It was observed that urea promoted angiogenesis in the CAM of duck embryos, especially at higher concentrations (P<0.05). Embryos treated with urea resulted in an alteration of the head-beak length (P<0.05). However, weight, crown-rump length, forelimb length, and hind limb length were not affected. The developing liver of urea-treated embryos showed distortion of the central vein shape and had larger sinusoidal spaces. The presence of Kupffer cells and lipid droplets were observed in the treated section. Congestion of blood cells, haemorrhage, and necrosis of hepatocytes were also observed in the tissue suggesting the extent of damage caused by the fertilizer. The findings of this study showed multiple developmental effects of urea on duck embryos. Further investigations are needed to shed more light on the toxicity of urea fertilizer on vertebrates.

Ammonia (NH₃) volatilized from soils plays an important role in N cycle and air pollution, thus it is important to trace the emission source and predict source contributions to development strategies mitigating the environmental harmful of soil NH₃ volatilization. The measurements of ¹⁵N natural abundance (δ¹⁵N) could be used as a complementary tool for apportioning emissions sources to resolve the contribution of multiple NH₃ emission sources to air NH₃ pollution. However, information of the changes of δ¹⁵N–NH₃ values during the whole volatilization process under different N application rates are currently lacking. Hence, to fill this gap, we conducted a 15-day incubation experiment included different urea-N application rates to determine δ¹⁵N values of NH₃ during volatilization process. Results showed that volatilization process depleted ¹⁵N in NH₃. The average δ¹⁵N value of NH₃ volatilized from the 0, 20, 180, and 360 kg N ha⁻¹ treatment was −16.2 ± 7.3‰, −26.0 ± 5.4‰, −34.8 ± 4.8‰, and −40.6 ± 5.7‰. Overall, δ¹⁵N–NH₃ values ranged from −46.0‰ to −4.7‰ during the whole volatilization process, with lower in higher urea-N application treatments than those in control. δ¹⁵N–NH₃ values during the NH₃ volatilization process were much lower than those of the primary sources, soil (−3.4 ± 0.1‰) and urea (−3.6 ± 0.1‰). Therefore, large isotopic fractionation may occur during soil volatilization process. Moreover, negative relationships between soil NH₄⁺-N and NH₃ volatilization rate and δ¹⁵N–NH₃ values were observed in this study. Our results could be used as evidences of NH₃ source apportionments and N cycle.

Extracellular Polymeric Substances (EPS) influenced Poly Cyclic Aromatic Hydrocarbons (PAHs) degrading Klebsiella pneumoniae was isolated from the marine environment. To increase the EPS production by Klebsiella pneumoniae, several physicochemical parameters were tweaked such as different carbon sources (arabinose, glucose, glycerol, lactose, lactic acid, mannitol, sodium acetate, starch, and sucrose at 20 g/L), nitrogen sources (ammonium chloride, ammonium sulphate, glycine, potassium nitrate, protease peptone and urea at 2 g/L), different pH, carbon/nitrogen ratio, temperature, and salt concentration were examined. Maximum EPS growth and biodegradation of Anthracene (74.31%), Acenaphthene (67.28%), Fluorene (62.48%), Naphthalene (57.84%), and mixed PAHs (55.85%) were obtained using optimized conditions such as glucose (10 g/L) as carbon source, potassium nitrate (2 g/L) as the nitrogen source at pH 8, growth temperature of 37 °C, 3% NaCl concentration and 72 h incubation period. The Klebsiella pneumoniae biofilm architecture was studied by confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM). The present study demonstrates the EPS influenced PAHs degradation of Klebsiella pneumoniae.

Bio-stimulation of the indigenous microbial community is considered as an effective strategy for the bioremediation of polluted environments. This examination explored the near effects of various bio-stimulants on pyrene degradation, prokaryotic community compositions, and functions using 16S rRNA amplicon sequencing and qPCR. At first, the results displayed significant differences (p < 0.05) between the prokaryotic community structures of the control group, PYR (contains pyrene only), and bio-stimulants amended groups. Among the bio-stimulants, biochar, oxalic acid, salicylate, NPK, and ammonium sulfate augmented the pyrene degradation potential of microbial communities. Moreover, the higher abundance of genera, such as Flavobacterium, Hydrogenophaga, Mycobacterium, Rhodococcus, Flavihumibacter, Pseudomonas, Novosphingobium, etc., across the treatments indicated that these genera play a vital role in pyrene metabolism. Based on the higher abundance of GP-RHD and nidA genes, we speculated that Gram-positive prokaryotic communities are more competent in pyrene dissipation than Gram-negative. Furthermore, the marked abundance of nifH, and pqqC genes in the NPK and SA treatments, respectively, suggested that different bio-stimulants might enrich certain bacterial assemblages. Besides, the significant distinctions (p < 0.05) between the bacterial consortia of HA (humic acid) and SA (sodium acetate) groups from NPK, OX (oxalic acid), UR (urea), NH4, and SC (salicylate) groups also suggested that different bio-stimulants might induce distinct ecological impacts influencing the succession of prokaryotic communities in distinct directions. This work provides new insight into the bacterial degradation of pyrene using the bio-stimulation technique. It suggests that it is equally important to investigate the community structure and functions along with studying their impacts on degradation when devising a bio-stimulation technology.

(S,S)-ethylenediaminedisuccinic acid (EDDS) has a strong capacity to mobilize potentially toxic elements (PTEs) in phytoextraction. It can release NH₄⁺-N via biodegradation, which can enhance N supply to soil thereafter promote plant growth and plant resistance to PTEs. However, the advanced feature of released N in the EDDS-enhanced phytoextraction remains unclear. In the current study, the effects of N supply released from EDDS on ryegrass phytoextraction and plant resistance to PTEs were investigated in detail by a comparison with urea. Our results supported that the addition of both EDDS and urea increased N concentration in soil solution, yet EDDS needed more time to release available N for plant uptake and transported more N from root to shoot. Additionally, EDDS significantly increased the concentration of all targeted PTEs, i.e. Cu, Zn, Cd, and Pb, in the soil solution, which results in higher levels of their occurrence in plant biomass compared with urea. By contrast, the supply of N slightly enhanced the ryegrass uptake of micro-nutrients, i.e. Cu and Zn, yet it caused negligible effects on nonessential elements, i.e. Cd and Pb. The mobilized PTEs by EDDS lead to elevated oxidative stress because higher levels of malondialdehyde and O₂•⁻ were observed. The supply of N attenuated oxidative stress caused by O₂•⁻ and H₂O₂, which was associated with enhanced activities of superoxide dismutase and peroxidase. Our results advanced the understanding of the exogenous N supply and metal resistance mechanisms in the EDDS-enhanced phytoextraction. This study also highlighted that EDDS can serve as a N source to ease N-deficient problems in PTEs-contaminated soils.

Nitrous oxide (N₂O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH₃ oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N₂O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N₂O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH₃ oxidizers and denitrifiers, including the previously-overlooked taxa, in N₂O emission from a cropland, and address the biological and environmental factors controlling the N₂O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N₂O production than the long-studied ones. We also demonstrated that the N₂O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N₂O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N₂O production by overlooked denitrifying bacteria and N₂O reduction by long-studied N₂O-reducing bacteria after manure fertilization into the plowed layer; and (3) N₂O production by NH₃-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH₃ oxidizers and denitrifiers, which will help us understand the environmental context-dependent N₂O emission processes.

Nitrogen (N) addition and mowing can significantly influence micronutrient cycling in grassland ecosystems. It remains largely unknown about how different forms of added N affect micronutrient status in plant-soil systems. We examined the effects of different N compounds of (NH₄)₂SO₄, NH₄NO₃, and urea with and without mowing on micronutrient Fe, Mn, Cu, and Zn in soil-plant systems in a meadow steppe. The results showed that (NH₄)₂SO₄ addition had a stronger negative effect on soil pH compared with NH₄NO₃ and urea, resulting in higher increases in soil available Fe and Mn herein. Nitrogen addition decreased plant community-level biomass weighted (hereafter referred to as community-level) Fe concentration but increased Mn concentration, with a greater effect under (NH₄)₂SO₄ addition. Community-level Cu concentration increased with (NH₄)₂SO₄ and NH₄NO₃ addition only under mowing treatment. Mowing synergistically interacted with urea addition to increase community-level Mn and Zn concentrations even with decreased soil organic matter, possibly because of compensatory plant growth and thus higher plant nutrient uptake intensity under mowing treatment. Overall, responses of plant-soil micronutrients to N addition varied with mowing and different N compounds, which were mainly regulated by soil physicochemical properties and plant growth. Different magnitude of micronutrient responses in plants and soils shed light on the necessity to consider the role of various N compounds in biogeochemical models when projecting the effects of N enrichment on grassland ecosystems.

Synthetic inhibitors and organic amendment have been proposed for mitigating greenhouse gas N₂O emissions. However, their combined effect on the N₂O emissions and ammonia-oxidizer (ammonia-oxidizing bacteria and archaea, AOB and AOA) communities remains unclear in calcareous soils under climate warming. We conducted two incubation experiments (25 and 35 °C) to examine how N₂O emissions and AOA and AOB communities responded to organic amendment (urea plus cattle manure, UCM), and in combination with urease (N-(n-butyl) thiophosphoric triamide, NBPT) and nitrification inhibitor (nitrapyrin). The treatments of UCM + nitrapyrin and UCM + nitrapyrin + NBPT significantly lowered total N₂O emissions by average 64.5 and 71.05% at 25 and 35 °C, respectively, compared with UCM treatment. AOB gene abundance and α-diversity (Chao1 and Shannon indices) were significantly increased by the application of urea and manure (P < 0.05). However, relative to UCM treatment, nitrapyrin addition treatments decreased AOB gene abundance and Chao 1 index by average 115.4 and 30.4% at 25 and 35 °C, respectively. PCA analysis showed that UCM or UCM plus nitrapyrin notably shifted AOB structure at both temperatures. However, fertilization had little effects on AOA community (P > 0.05). Potential nitrification rate (PNR) was greatly decreased by nitrapyrin addition, and PNR significantly positively correlated with AOB gene abundance (P = 0.0179 at 25 °C and P = 0.0029 at 35 °C) rather than AOA (P > 0.05). Structural equation model analysis showed that temperature directly increased AOA abundance but decrease AOB abundance, while fertilization indirectly influenced AOB community by altering soil NH₄⁺, pH and SOC. In conclusion, the combined application of organic amendment, NBPT and nitrapyrin significantly lowered N₂O emissions via reducing AOB community in calcareous soil even at high temperature. Our findings provide a solid theoretical basis in mitigating N₂O emissions from calcareous soil under climate warming.

Potato (Solanum tuberosum L.) production in irrigated coarse-textured soils requires intensive nitrogen (N) fertilization which may increase reactive N losses. Biological soil additives including N-fixing microbes (NFM) have been promoted as a means to increase crop N use efficiency, though few field studies have evaluated their effects, and none have examined the combined use of NFM with microbial inhibitors. A 2-year study (2018–19) in an irrigated loamy sand quantified the effects of the urease inhibitor NBPT, the nitrification inhibitor DMPSA, NFM, and the additive combinations DMPSA + NBPT and DMPSA + NFM on potato performance and growing season nitrous oxide (N₂O) emissions and nitrate (NO₃⁻) leaching. All treatments, except a zero-N control, received diammonium phosphate at 45 kg N ha⁻¹ and split applied urea at 280 kg N ha⁻¹. Compared with urea alone, DMPSA + NBPT reduced NO₃⁻ leaching and N₂O emissions by 25% and 62%, respectively, and increased crop N uptake by 19% in one year, although none of the additive treatments increased tuber yields. The DMPSA and DMPSA + NBPT treatments had greater soil ammonium concentration, and all DMPSA-containing treatments consistently reduced N₂O emissions, compared to urea-only. Use of NBPT by itself reduced NO₃⁻ leaching by 21% across growing seasons and N₂O emissions by 37% in 2018 relative to urea-only. In contrast to the inhibitors, NFM by itself increased N₂O by 23% in 2019; however, co-applying DMPSA with NFM reduced N₂O emissions by ≥ 50% compared to urea alone. These results demonstrate that DMPSA can mitigate N₂O emissions in potato production systems and that DMPSA + NBPT can reduce both N₂O and NO₃⁻ losses and increase the N supply for crop uptake. This is the first study to show that combining a nitrification inhibitor with NFM can result in decreased N₂O emissions in contrast to unintended increases in N₂O emissions that can occur when NFM is applied by itself.

Urea is one of the most commonly used nitrogen fertilizers in agricultural soil and is easily decomposed by soil urease resulting in ammonium release. The produced ammonium can be volatilized or converted to nitrate, which is susceptible to leaching, leading to groundwater contamination unless used by plants. Hence, it is important to control the release of nitrogen from the urea. Pyroligneous acid inhibited the urease activity and decreased ammonium release up to 80% compared to the control. Amidase including asparaginase and glutaminase is an enzyme that catalyzes hydrolysis of amide group, similar to urease. Therefore, the effect of pyroligneous acid on the inhibition of soil amidase was also tested and the results showed that pyroligneous acid competitively inhibited asparaginase while glutaminase was not inhibited. However, inhibitory effect of pyroligneous acid on asparaginase was negligible compared to the urease. The application of pyroligneous acid with a smaller amount of urea for controlled nitrogen release during Chinese cabbage growth showed that dry biomass and nutrient contents of Chinese cabbage were similar to the case of the conventional urea application. The nitrogen utilization efficiency (NUE) was highest for 33% less amount of urea supply with pyroligneous acid (2.21) compared to conventional treatment (1.81). Consequently, the use of pyroligneous acid with urea enhances nitrogen use efficiency while also protecting environments from non-point source contamination.