Humate-Coated Urea as a Tool to Decrease Nitrogen Losses in Soil

Abstract

Processes of N transformation in soil as affected by application of the three kinds of urea fertilizers, conventional urea (U), humate-coated urea (U_HA), and urea treated with the urease inhibitor NBPT (U_UI), are examined in a model laboratory experiment. Effects of urea fertilizers on soil chemical (content of water-extractable N-NH₄ and N-NO₃), and microbiological properties (rate of actual and potential N₂O emission, basal and substrate-induced respiration, microbial biomass C, emission of ethylene) are focused to answer the following questions: (i) whether humate-coated urea has the ability to decrease N losses in soil; and (ii) how it affects soil biological activity comparable to synthetic urease inhibitor. The results showed that U_HA demonstrated advantages comparable to U in its ability to decrease N losses in soil: it increased N-NH₄ content by 35%, reduced nitrate content by 9%, and decreased N₂O emissions by 50%. U_HA promoted basal soil respiration by 10% and the specific activity of the soil microbial community by 7%, providing the highest metabolic quotient qCO₂. Comparably to NBPT-treated U, U_HA mainly shows intermediate results between U-UI and conventional U. Considering the low cost of raw humates, U-HA can be regarded as a promising tool to decrease N losses in soils.

1. Introduction

Nitrogen is the essential element for plant nutrition and an optimum supply of N is the key factor for the agricultural production. Globally, application of N fertilizers increased from 32 million tons per year in 1970 and is expected to achieve 130–150 million tons per year by 2050 [1]. However, the proper management of N supply in the soil–plant system is challenging due to unproductive losses of nitrogen due to volatilization (gaseous losses in the form of ammonia and nitrogen oxides) and leaching as nitrate ions. In modern agricultural systems, the N losses can equal up to 50–70% of applied N [2,3,4]. In turn, it leads to the increase in NH₃ and N₂O concentrations in the Earth’s atmosphere, which contribute to greenhouse gas-driven climate change. Leaching of nitrates (NO₃⁻) from terrestrial to aquatic ecosystems causes changes to ecosystem productivity and biological diversity, eutrophication, and nitrate contamination of freshwater resources.

Urea is the predominant form of N fertilizers and contributes to more than half of the global agricultural N input [5]. Thus, it is characterized by both the largest volumes of application and large unproductive losses of nitrogen. Chemically, urea is carbonic acid diamide (NH₂)₂CO containing 46% N. Transformation of urea in soil is described elsewhere [6,7,8]. In brief, in soil, it quickly undergoes ammonification: the hydrolysis is catalyzed by soil enzyme urease secreted by microorganisms, and it decomposes with the formation of gaseous ammonia and CO₂, which volatilize from the soil. In most agricultural soils, ammonification occurs within a few days, depending on soil moisture, temperature, and biological activity.

Partially, urea also dissolves in soil water and is converted into ammonium carbonate. In turn, the formed ammonium is partially immobilized by organisms (plants and bacteria) and fixed by soil organomineral components, and it is partially subjected to nitrification (oxidation of NH₄⁺ to NO₃⁻), catalyzed by diverse soil microorganisms. As a result, nitrites and nitrates are formed and serve as a source of N for plant nutrition. Nitrates, besides being assimilated by plants and accumulated in agricultural products, are poorly soil-bound anions and easily leach and run off into groundwater, ultimately causing eutrophication of water resources. Once in anaerobic conditions (for example, inside soil aggregates), nitrates are exposed to a set of denitrifying soil microorganisms, which reduce them to N₂, N₂O, and other nitrogen-containing gases. Denitrification is another cause of losses of fertilizer-derived N.

One of the contemporary tools to mitigate nitrogen losses is the use of synthetic inhibitors of the nitrogen cycle processes: inhibitors of nitrification, urease or others. Basically, such inhibitors are physiologically active compounds that interact with protein molecules of key enzymes [9,10,11]. They integrate into their structure, either disrupting the functioning of the active site or causing complete denaturation of the protein. Thus, they inhibit the action of enzymes and reduce the formation of those nitrogen compounds that are easily removed from the soil: leached (as nitrate) or volatilized as gases (ammonia, nitrogen and its oxides). However, in spite of synthetic inhibitors’ ability to increase nitrogen’s usage efficiency, there are many drawbacks, including difficulties in application, cost, degradation, pollution, and entry into the food system [12,13]. Thus, inhibitors provoke more or less serious disruption and imbalance of the natural nitrogen cycle. Many of these compounds exhibit nonspecific activity and are capable of disrupting the action of a number of enzymes through various mechanisms [14] and there is evidence of their negative impact on the food production quality [15,16].

As an alternative, one can consider the use of inhibitors of natural origin [10]. Among them are humic substances (HSs), macromolecular natural organic matter consisting of a negatively charged polyelectrolyte that contains a hydrophobic core and a variety of functional groups, including quinone, aldehyde, carboxyl, phenolic and alcoholic hydroxyls, and ether [17].

They may act as potential urease inhibitors. Possible mechanisms of their action include chemical (ammonium fixation due to interaction with functional groups of HSs) and biochemical processes (enzyme inhibition by polyphenolic components of HSs) [18]. HSs are able to form strong complexes with oppositely charged proteins, which lead to changes in the enzyme activity. Li et al. [19] reported that HSs affected the activity and stability of urease depending on pH, ionic strength, and mass ratio HS/enzyme. Liu et al. [20] showed the inhibition of the urease activity by HSs and hypothesized that mechanisms of inhibition may be an interaction of the functional groups of HSs with the thiol group of urease and the formation a larger particle size of the complex to inhibit the activity of urease. Finally, due to the presence of numerous acidic functional groups, conditioning a high exchange capacity, HSs facilitate abiotic fixation of ammonia, which in turn decreases ammonia volatilization and mitigates NO₃⁻ leaching and N₂O emissions, thus reducing nitrogen loss [21,22]. Moreover, a big advantage of the use of HSs is the fact that they can be produced at a low cost from a low-rank coal and they are available on the market.

Moreover, the combination of well-known beneficial properties of HSs and inorganic fertilizers (urea) provides more balanced nutrition for crops and optimization of soil properties. Encapsulation of mineral fertilizer granules in an HS coating provides certain benefits comparably to conventional N fertilizers. Such organomineral granules supply organic matter together with added nutrients, thus slowing down immediate nutrient release due to increased adsorption by organic matter and/or microbial nutrient immobilization. An HS coating reduces the dissolution rate of the urea granules and, accordingly, slows down the contact with urease. In the further processes of N transformations, the presence of HSs in the interphases with soil microbial community can contribute to population density of soil ammonia oxidizers [23], promote ammonia fixation, and prevent the nitrate formation with subsequent leaching and/or volatilization as nitrogen oxides and ammonia [24,25,26]. Positive effects of HS urea fertilizers on crop yield are also reported [27,28,29,30,31,32,33].

However, the intensity of the manifestation of these processes depends both on the soil and climatic conditions and on the properties of the humates themselves. In regions with arid, hot climates, where the majority of rainfall occurs only in spring, there are large gaseous losses of N fertilizer volatilized as ammonia. This encourages farmers to use increased doses of nitrogen fertilizers, which leads to both higher financial costs and accelerated SOM mineralization, soil depletion, and environmental pollution.

On the other hand, in regions with a humid climate and high precipitation, nitrogen losses are high due to nitrate leaching and N₂O emissions, which are also accompanied by the removal of soil organic matter. These processes also provoke farmers to use increased doses of nitrogen fertilizers, which in turn leads to soil dehumification greenhouse gas emissions and overall environmental pollution. Therefore, both in arid regions (such as the Mediterranean, Central Asia, and the Middle East) and humid regions (like Thailand, Indonesia, Malaysia, southern India, and China), the use of humate-coated urea as a “green” alternative to traditional chemicals seems especially promising.

Moreover, HSs themselves significantly vary in chemical properties and biological activity depending on their organic matter origin (lignite, brown coal, leonardite, peat, organic shale, compost, etc.) [34,35]. Therefore, a pilot sample of a humate-coated fertilizer requires preliminary testing in appropriate soil conditions to draw a conclusion on its effectiveness and prospects of use.

The objective of this study was to examine the processes of N transformation in soil as affected by the application of the three kinds of urea fertilizers: humate-coated urea, urea treated with urease inhibitor, and conventional urea. The study was conducted in a model laboratory experiment simulating spring waterlogging of the soil to provoke maximal leaching of nitrates. Effects of urea fertilizers on soil chemical (content of water-extractable N-NH₄ and N-NO₃) and microbiological properties (rate of actual and potential N₂O emission, basal and substrate-induced respiration, microbial biomass C, emission of ethylene) are focused to answer the following questions: (i) whether humate-coated urea has advantages comparable to conventional urea in its ability to decrease N losses in soil; and (ii) how it affects soil biological activity comparable to synthetic urease inhibitor.

2. Materials and Methods

2.1. Materials

Conventional urea (U), containing 46% N, was purchased from a local producer and used as received. Humate-coated urea (U_HA) was produced by Life Force Group LLC (Moscow, Russia) and prepared as follows. Urea granules were placed in a mixer tank and treated with liquid 30% wt potassium humate containing 20% of HS (humic and fulvic acids). Liquid potassium humate was produced by the Life Force Group LLC (Russia) by alkaline extraction from leonardite, which originated in the Krasnoyarsk region (Russia). The humate solution is fed through the nozzle, and then a stream of heated air (85 °C) is supplied with constant stirring. During the following cooling and drying, the applied potassium humate acts as an anticaking agent and organic adhesive, sticking to the urea granules. The final U_HA product is urea granules encapsulated in a shell of potassium humate and containing 2% HA.

To compare the effect of U_HA on the reduction in ammonia volatilization, a separate sample of U was treated with a urease inhibitor (UI), N-(n-butyl) thiophosphoric triamide (NBPT) (Rhodia Operations, Paris, France), following the recommendations provided by the manufacturer using the application rate of 3.2 L per 1000 kg of urea. To obtain UI-treated urea (U_UI), 4 g of U was placed in a flask, 12.8 µL of NBPT was added, and it was thoroughly mixed by shaking by hand.

The soil used was the top horizon of cultivated sandy loam Abruptic Luvisol (7.5% clay, 30.5% silt, and 62.0% sand), sampled in the Kaluga region of Russia (54.4293 N, 36.5353 E) with pH 6.3, 1.76% of total C, and 0.18% of total N.

2.2. Experiment

A soil sample with natural moisture content (11.7%) was kneaded, and large roots were removed. Fresh soil was sieved through a 3 mm sieve and stored in a refrigerator until the start of the experiment (four days). Before setting the experiment, the soil was kept for 2 h at room temperature. Samples of fresh undried soil (200 g) were placed in 300 mL pots with the holes in the bottom. A nylon filter was preliminarily placed on the bottom of each pot. In parallel, we took soil samples to determine moisture content to bring the results to a moist-free basis. Each treatment was compiled in 5 parallels.

Experimental treatments included control soil (Control), and soil treated with three kinds of urea: conventional urea (U), humate-coated urea (U_HA), and conventional urea treated with urease inhibitor NBPT (U_UI). Urea fertilizers were applied at a specific rate, giving 0.1803 g of N/kg, as recommended for many agricultural crops. Thus, the weights of U and U_UI (46% of N) were 0.0780 g/pot and 0.0795 g/pot of U_HA (2% of HA and 98% of urea). Granules of fertilizers were uniformly placed on the soil surface in pots. To mimic the conditions of spring waterlogging, a volume of distilled water slightly exceeding the soil water holding capacity (70 mL) was poured onto the soil surface to provide complete water penetration and drops were collected on the drip pan.

The pots were placed in a climate chamber Binder KBW400 and exposed at 22 °C and constant illumination for 21 days. On the 5th, 8th, and 15th days of exposure, the soils were additionally moistened with a volume of water simulating a heavy rain. According to the Glossary of Meteorology [36], rainfall is classified as a heavy rain when the precipitation rate is between 7.6 and 50 mm per hour. We simulated 10 mm of precipitation, carefully adding 50 mL of distilled water. The appearance of drops on the drip pan was recorded.

On day 22, the soils were removed from the pots, homogenized, and individual samples were taken from each pot for moisture determination, chemical, and microbiological analysis.

2.3. Chemical Analyses

The content of nitrate and ammonium was determined in water extracts (1:2.5) using the multichannel ion meter Expert-001 with ion-selective electrodes (ISEs) ECOM-NH₄ and ECOM-NO₃. Prior to measurements of the activity of nitrates and ammonium ions, solutions of background electrolytes were added to the test extracts in order to control the ionic strength of the solutions (0.1 N K₂SO₄ for nitrates and 0.1 N NaNO₃ for ammonium) as recommended by the ISEs’ manuals. The obtained values of ammonium and nitrate content were recalculated for N content. The pH values were determined in the same extracts using a glass electrode. For analysis, samples of initial moisture content were used, and the results were calculated on a moist-free basis. Soil moisture was determined gravimetrically.

2.4. Analysis of the Intensity of the Nitrogen and Carbon Cycle Processes

After the end of the exposure, and prior to sampling for chemical analyses, soil samples (5 g) were taken with a core sampler and placed in 15 mL glass vials. To determine soil basal respiration (BR) and actual losses of nitrogen as N₂O, vials were closed with rubber caps and incubated for 24 h in a thermostat at 22 °C. After that, from each vial, a gas sample (0.25 mL) was taken with a syringe, and concentrations of CO₂, C₂H₄, and N₂O were determined on a gas chromatograph Crystal 5000.2 (Chromatek, Yoshkar-Ola, Russia), as described below.

To determine substrate-induced respiration (SIR), 0.5 mL of glucose solution at a rate giving 2.5 mg of glucose per 1 g of soil was additionally added to the samples. Then, the vials were closed with rubber stoppers and incubated for 2–3 h in a thermostat at 22 °C. The incubation time of each sample was registered and used in the calculations, and the initial CO₂ content in the laboratory air was also considered. The SIR value is proportional to the carbon of microbial biomass because, during the measurement, all the biomass of heterotrophic aerobic microorganisms is activated by the available organic substrate.

The intensity of N₂O emission was measured using the acetylene method, in which acetylene acts as an inhibitor of nitrous oxide reductase [37]. In the presence of acetylene C₂H₂, the final product of denitrification is N₂O, which can be determined with high accuracy on a gas chromatograph.

To determine potential N₂O emission, KNO₃ (0.3 mg/g) and glucose (2.5 mg/g) were added to soil samples in vials, closed with rubber caps with aluminum clips, and purged with argon for 2 min to remove oxygen. After that, 1 mL of acetylene was added to each vial using a syringe, and then they were vigorously shaken by hand to distribute acetylene throughout the soil volume. The vials were incubated for 24 h in a thermostat at 22 °C, then a gas sample (0.25 mL) was taken with a syringe. In gas samples, the concentration of N₂O was determined using a gas chromatograph Crystal 5000.2 (Chromatek, Russia).

The gas chromatograph is supplied with two metal chromatographic columns 2 m long and 1 mm in inner diameter, filled with Hayesep-D 80/100. A thermal conductivity detector (TCD) and a flame ionization detector (FID) are connected in chain to one of the columns to provide the measurement of CO₂, CH₄, C₂H₄, and other volatile hydrocarbons; the carrier gas is helium. The second column is connected to an electron capture detector (ECD) on which N₂O is measured; the carrier gas is nitrogen.

Computation of Additional Parameters of the State of Soil Microbial Communities

Carbon of microbial biomass (Cmic) was calculated as following [38]:

Cmic (µg C g⁻¹) = SIR (µlCO₂ g⁻¹ h⁻¹) • 40.04 + 0.37

(1)

To estimate the specific activity of the soil microbial community, the metabolic quotient (qCO₂) was calculated as the ratio of BR to Cmic and expressed in mg CO₂ µg⁻¹ Cmic h⁻¹. The metabolic quotient is a measure of the fundamental physiological state of the microbial community and shows the amount of carbon that is formed per unit of microbial biomass per hour.

2.5. Statistics

All the measurements were carried out in five repetitions. Statistica 10 software package was used for the statistical analyses. All the reported values are given as mean ± SD. For each group of analyses, to determine the significance of differences, the differences were compared using one-way analysis of variance (ANOVA). Fisher’s LSD test was employed to identify differences between treatments. All tests of significance were carried out at p < 0.05.

3. Results

3.1. Content of N-NH₄, N-NO₃, and Soil pH

The initial untreated soil is very poor in ammonium (less than 1 ppm N-NH₄), and the application of 180 ppm of N with urea fertilizers drastically increased the content of water-soluble N-NH₄ in the soil (Figure 1a). Conventional urea increased N-NH₄ content up to 2.3 mg/kg and humate-coated urea up to 3.5 mg/kg. The maximum accumulation of ammonium, as expected, was observed in the treatment using a urease inhibitor. By inhibiting the action of the enzyme, UI reduces the decomposition of urea and, accordingly, the loss of nitrogen in the form of gaseous NH₃. As a result, nitrogen in urea is more fully converted into NH₄⁺ and binds with soil components.

Due to the intensive nitrification process, occurring during the experiment, the majority of applied N was converted into nitrates (see

Table 1. Effect of urea fertilizers on content of N-NH₄, N-NO₃ (normalized by the amount of applied N), and actual N₂O emission rate (as % of U-treated soil); p < 0.05.

Treatment	N-NH4	N-NO3	N-N2O, %
Control	NA 1	NA 1	9.6
U	1.25	97.2	100.0
U_HA	1.93	88.2	50.1
U_UI	2.34	85.5	89.7

¹ NA—not applicable.

). In absolute values, the content of N-NO₃ in water extracts was the highest in the treatment with conventional urea and lower in row U_HA–U_UI (Figure 1b). According to the LSD test, the lower accumulation of nitrates in the treatments with HA and UI compared to conventional urea is statistically confirmed, whereas values for U_HA and U_UI do not differ between each other.

Active transformation of N-NH₄⁺ into nitrates by nitrifying microorganisms resulted in a decrease in soil pH by 0.2–0.5 units in all treatments with urea compared to control (Figure 1c). This effect was most clearly manifested in the treatment with conventional urea, while the use of U_HA and especially U_UI contributed to a lower increase in soil acidity.

3.2. Gaseous N Losses

In this experiment, processes of ammonification and nitrification in soil were intensively manifested, and a large share of applied N accumulated as nitrates. Therefore, the most significant source of N losses is the emission of N₂O caused by the activity of microbial denitrifiers.

Application of urea is expectedly followed by a significant increase in gaseous losses of nitrogen in the form of nitrogen monoxide N₂O (Figure 2a). Among all the urea-treated samples, the lowest N₂O emission was observed for the U_HA treatment: 50% less compared to conventional urea, and the differences are statistically significant.

As for potential denitrification activity, all urea fertilizers decreased it by about 30% (Figure 2b). This is the result of changes in soil conditions and the pattern of microbial succession over the 22 days of the experiment. Because the differences between U_HA, U_UI, and conventional U are not observed, one can conclude that even under conditions most favorable for denitrification, humate-coated U does not provoke higher emissions of N₂O (a greenhouse gas) into the atmosphere. Under natural conditions, actual N₂O emissions are likely to be significantly lower than when using conventional U or urea in combination with urease inhibitors.

3.3. Activity of Soil Microbial Community

Application of mineral fertilizers inevitably entails profound changes in soil biological activity level, which affects the processes and rate of biochemical transformation of biophilic elements. In this experiment, application of all the tested urea fertilizers led to a decrease in carbon dioxide emissions (basal respiration) from 468 in the control treatment to 343–395 ng C-CO₂ g⁻¹ h⁻¹ (Figure 2a). Conventional U provided the highest manifestation of this effect, whereas the presence of a urease inhibitor and humate coating slightly mitigated this effect.

The decrease in BR is also related to a decrease in soil pH, which occurs with urea application (Figure 1c). The highest increase in acidity level was observed in the treatment with U. In turn, this also caused the most significant decrease in BR, while with the application of U_HA and especially U_UI, microbial activity was less suppressed. As a result, the activity of the soil microbial community (expressed as BR) in U_HA was higher compared to conventional U and the same as in U_UI.

The amount of soil microbial biomass C was lower in all urea-treated samples compared to the control (Figure 3b), most probably as a result of increased competition for available carbon sources while providing nitrogen nutrition.

In addition to the biomass itself, the microbial metabolic quotient (respiration-to-biomass ratio), or qCO₂, is an important qualitative characteristic that indicates the state and specific activity of the soil microbial community. In our experiment, the qCO₂ values ranged within 0.72–0.77 µg CO₂ mg⁻¹ Cmic h⁻¹ (Figure 3c). Although the differences between treatments are not statistically significant, at a trend level, a decrease in qCO₂ as affected by conventional U and U_UI is noticeable. On the other hand, with the application of humate-coated urea, the level of functional activity of microbial biomass is preserved at the level of control values.

The initial soil is characterized by ethylene emission at a level of 40 ng C-C₂H₄ g⁻¹ h⁻¹ (Figure 3d). Ethylene is known as one of the most important phytohormones, and even at low concentrations, it has a significant impact on plant growth and development. According to Arshad and Frankenberger [39], soil fungi and bacteria also produce enough C₂H₄ to trigger physiological reactions. However, little is known about the particular mechanisms underlying the ethylene-mediated interaction of soil microorganisms and plants. While ammonium forms of N do not affect or even enhance ethylene synthesis in soils, nitrates are known to have a detrimental impact on it [40]. In our experiment, we did not observe the dependence of ethylene emission on the concentration of nitrates; however, it should be mentioned that under the influence of humate-coated urea and a urease inhibitor, ethylene emission is reduced by half compared to the control.

4. Discussion

In this study, we examined the transformation of nitrogen in soil as affected by the three kinds of urea fertilizers: conventional U, humate-coated U, and U in combination with a urease inhibitor. Under the given experimental conditions, accumulation of N-NH₄ and a decrease in N-NO₃ formation were observed in the latter two treatments comparable to conventional urea.

The presentation of the obtained data as a percentage of applied N gives a clearer understanding of the occurring processes (Table 1). Ammonia fixation is promoted in the presence of HA coating, as evidenced by the increase in the content of water-extractable NH₄⁺. Although this effect is less manifested compared to the treatment with a urease inhibitor, it definitely shows a positive trend. Such a mechanism may be due to the nature of chemical bonds, mainly carboxylic and phenolic groups located in the peripheral part of humic acid, which promote the binding of cations and thus fix them in soil.

In soil, urea rapidly undergoes the process of nitrification with the formation of nitrate ions. The nitrification rate depends on soil moisture, the content of nutrients, and soil biological activity. In our experiment, the initial soil conditions were favorable for nitrification, and 86–97% of the applied N was converted into nitrates (Table 1). The highest share of nitrates was detected in the treatment with conventional urea (97%), whereas in the treatment with U_HA, the share of nitrates was close to that for the treatment with the urease inhibitor (88% and 86%, respectively). This fact indicates a decrease in the nitrification rate under the influence of humate-coated U.

Active nitrification could be the main reason for the decrease in soil pH in the treatment with urea alone (Figure 1c). Normally, in the first days after urea application, soil pH rises due to the formation of an alkaline salt, ammonium carbonate (NH₄)₂CO₃. Later, however, as a result of ammonium’s nitrification and the subsequent formation of nitrites and nitrates, alkalization is replaced by acidification. As one can see from the above, the presence of HA decreases the nitrification rate and thus mitigates soil acidification. In turn, less acidity is more favorable for maintaining soil biological activity level, which is evidenced by higher values of BR (

Table 2. Effect of urea fertilizers on soil microbiological parameters, % to control, p < 0.05.

Treatment	BR	SIR	Cmic	Ethylene
Control	100.0	100.0	100.0	100.0
U	76.8	81.7	81.8	75.3
U_HA	85.1	79.0	87.3	46.9
U_UI	81.0	87.2	79.0	40.9

).

For a clearer understanding of the actual N₂O emission rate, we accepted the emission value from the treatment with U (0.145 ng N-N₂O g⁻¹ h⁻¹, Figure 2a) as 100%. (Table 1). One can see that N₂O emission drastically decreased in the U_HA treatment compared to conventional U. Gaseous N losses achieved the minimum value, being even less than in the treatment with the urease inhibitor. A similar situation will be observed for most of the natural conditions. This effect may be associated with the high hydrophobic properties of humic acids [41], which cover the urea granules and, thus, reduce the rate of urea dissolution and the availability of nitrogen for subsequent microbial transformation by communities of nitrifying and denitrifying prokaryotes in the soil.

All the above supports the hypothesis that the HA-coating of urea acts similar to a natural nitrification inhibitor. We consider it a tendency to stabilize the pool of applied ammonium and reduce the rate of its oxidation to nitrates, followed by the removal of the latter with soil runoff and gaseous losses. Under the conditions of our experiment, this pattern is expressed as a trend. One can assume that in soils with less biological activity, such as sandy or low-humus soils, this pattern may be more clearly manifested.

Moreover, the concentration of HA on urea granules also matters, because the urease activity inhibition rate is positively correlated with the proportion of added HA. According to the published data, the optimal proportion of HA in humic-enhanced urea may vary from 0.2 to 15% [42,43,44,45]. Some studies showed that urea blended with brown coal rich in HA can minimize mineral N losses by increasing the proportion of brown coal up to 65% [46,47]. At the same time, one should bear in mind that HA exhibits stimulatory effects on plant growth at 50–500 mg kg⁻¹ but inhibitory effects at 1000–4000 mg kg⁻¹ [34,48,49]; therefore, an optimal rate of HS on urea should be examined.

All the tested urea fertilizers also affected microbial transformation of carbon in soil. Basal microbial respiration decreased by 14–23% compared to the control (Table 2). These changes should be associated with a decrease in microbial biomass associated with successional changes in the composition of the microbial community as well as increased competition for readily available organic substrates. Biomass measurements have been used to give an early indication of the changes in the organic matter content of soils due to variation in soil management [50,51]. Humate-coated U can be considered gentler on the functioning of microbial communities and favorable for the stabilization of soil organic matter, because, in this treatment, there was a smaller decrease in biomass and basal respiration, as well as no change in the metabolic quotient compared to the control. In further studies, attention should also be paid to the enhanced suppression of the rate of ethylene emission from the soil with the use of humate-coated U and a urease inhibitor. Although ethylene is not a greenhouse gas, it is an important physiologically active substance, and the increase in its concentration in the pore space of the soil and the near-surface layer of the atmosphere can affect the growth and development of cultivated plants and, ultimately, yields.

5. Concluding Remarks

In our experiment, humate-coated urea demonstrated certain advantages compared to conventional urea in its ability to decrease N losses in soil. It promoted abiotic fixation of ammonia, prevented urea-caused soil acidification, and decreased nitrification and N₂O emissions. The microbial biomass also remains more stable, and its functioning is less disturbed.

It is noteworthy that these effects, besides having agricultural merit, are also of environmental importance. Emissions of ammonia and nitrogen monoxide lead not only to unproductive losses of N fertilizer but also entail unfavorable environmental consequences, contributing to the global greenhouse effect.

Comparably to NBPT-treated U, humate-coated U mainly shows intermediate results between U-UI and conventional U. At the same time, some indicators of soil biological activity showed the potential negative impact of the NBPT on soil biota. Thus, the lower metabolic quotient qCO₂ compared with U-HA and control soil indicates a possible nonspecific negative effect of the urease inhibitor on other functions of the soil biota. Considering the fact that the effects of U-HA follow the same trend as those of U_UI, and given the low cost of raw humates, its use can be economically feasible.

However, it is necessary to consider that the observed trends are valid for the conditions of this given experiment. The cultivated Abruptic Luvisol used in our experiment was characterized by a high level of biological activity and a neutral pH; and incubation was carried out at 22 °C under constant illumination. One can assume that under other experimental conditions, with other concentrations of HA in urea or in other soil and bioclimatic conditions, the revealed patterns may be more pronounced. Further research on this issue for various soils is worth implementing. Thus, in arid regions with low-humus sandy soils and intensive volatilization of NH₃, a humic coating on the surface of urea granules can not only act as a urease inhibitor but also improve soil humus state. In humid regions subjected to intensive nitrate leaching and active N₂O emission, humate-coated urea will also contribute to reducing the manifestation of these negative effects. Despite the fact that its effect is less pronounced compared to a synthetic urease inhibitor, the use of such products can be considered a tool to reduce chemical loads in agriculture and a step towards “green” farming.