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Article

Changes in Microbial Activity Associated with the Nitrogen Biogeochemical Cycle in Differently Managed Soils, Including Protected Areas and Those Reclaimed with Gangue

by
Jolanta Joniec
1,
Edyta Kwiatkowska
1,*,
Anna Walkiewicz
2 and
Grzegorz Grzywaczewski
3
1
Department of Environmental Microbiology, Faculty of Agrobioengineering, University of Life Sciences in Lublin, Leszczyńskiego 7, 20-069 Lublin, Poland
2
Department of Natural Environment Biogeochemistry, Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland
3
Department of Zoology and Animal Ecology, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(10), 4343; https://doi.org/10.3390/su17104343
Submission received: 1 April 2025 / Revised: 2 May 2025 / Accepted: 9 May 2025 / Published: 11 May 2025
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Abstract

The proximity of ecologically valuable areas to industrial zones indicates a strong need for monitoring their condition. Soil assessment involves both molecular techniques for studying microbial biodiversity, such as PCR, sequencing, and metagenomics, as well as parameters of biochemical and enzymatic activity of soil microorganisms. The authors studied the activity of microorganisms responsible for the nitrogen cycle to compare the condition of soils under different uses (wastelands and arable fields) located in the ecologically valuable areas of the Polesie National Park (PNP, protected area) and its surroundings. Additionally, they assessed the suitability of gangue for reclamation and its effectiveness depending on treatment duration (2 and 10 years). In most of the activities analyzed, their levels were lower in the park. A higher intensity of ammonification and nitrification was observed in the soil sampled from the field in the park; however, a reduced N2O emission was also recorded after incubation in the lab of soil samples collected in the autumn, which may indicate that nitrogen loss from the soil does not occur in this particular habitat, which requires further, long-term and cyclical field trials. These observations confirm the potential protective role of the park in relation to soils and atmosphere in the context of the nitrogen cycle. The activities under study in the reclaimed soils were in both cases lower than in soils from the fields. The current results prove that this method of reclamation is not entirely effective; however, long-term reclamation yielded better results. The present study provided valuable information on the effectiveness of the protective role of the PNP in relation to soils and air. Additionally, these results may be helpful in making decisions regarding the use of waste, such as gangue, for reclamation.

1. Introduction

Soil is one of the most important natural resources in the world, playing a crucial role in maintaining ecosystem balance and meeting basic human needs. At the same time, soil degradation, resulting from various anthropogenic and natural factors, is becoming an increasingly common phenomenon, threatening environmental stability and agricultural production [1]. The Food and Agriculture Organization of the United Nations (FAO) defines soil degradation as “a change in the soil health status resulting in a diminished capacity of the ecosystem to provide goods and services for its beneficiaries. Degraded soils have a health status such that they do not provide the normal goods and services of the particular soil in its ecosystem” [2]. It is a dynamic and complex process (encompassing past, present, and future degradation processes) caused by physical, chemical, and biological factors that frequently interact [1]. Land degradation, whether resulting from natural processes or human activities, is a continuous and unavoidable phenomenon that poses a significant threat to achieving the vision of the 17 Sustainable Development Goals of the United Nations [3].
One of the threats to environmental health, including soil health, is various types of waste, which, when improperly managed, contribute to soil degradation, as well as water and air contamination. Mining is one of the economic sectors that generates significant amounts of waste, including gangue [4,5].
The latter is a waste material generated during the extraction and processing of various minerals, including coal [5]. It is most commonly stored in massive heaps surrounding mines, significantly impacting not only the immediate environment but also regional ecology, posing a serious challenge for its management [6,7]. Among other things, it can contribute to soil structure damage, erosion and nutrient depletion, and can negatively affect the abundance and diversity of soil microbial communities [6,7,8,9]. In recent years, there has been a shift in the economic approach to gangue, which is increasingly regarded as a resource for mineral raw materials for various economic applications rather than merely as waste [6,7,10,11]. The processing and potential utilization of gangue are influenced by its physical and chemical properties, including chemical and granulometric composition, mechanical properties, carbon and other element contents, and moisture levels. When exploring potential applications for gangue, it is also essential to consider its environmental impact, including the presence of heavy metals and the potential leaching of salts such as sulfates and chlorides [6,7]. The assessment of the potential use of waste derived from coal mining or gangue-based products for reclamation purposes should be a comprehensive issue, addressing both regulations related to soil and water environmental quality, and local conditions [12].
Reclamation of degraded areas caused by industrial activity, particularly mining, should be given priority. However, it is also crucial to monitor the impact of mining activities on neighboring areas. The Polesie National Park in eastern Poland, located approximately 10 km from the Bogdanka coal mine, is an example of an interesting vicinity, due to the richness and uniqueness of its flora and fauna, which should be continuously monitored.
The Bogdanka coal mine is the only coal mine in Poland located outside the Upper Silesia region and the sole mine extracting coal in the Lublin Coal Basin (LCB). The Lublin Coal Basin (LCB) contains coal deposits and, importantly, is an area of diverse and highly valuable natural landscape and cultural features, a fact strongly emphasized on the mine’s official website [13]. The entire mining infrastructure, along with the mining sites, is surrounded by protected areas. The mining fields are adjacent to ecologically valuable areas such as the Uściwierz Lakes Natura 2000 site PLH-06009, Polesie Natura 2000 site PLB-060019, Łęczna Lake District Landscape Park, Polesie Protected Landscape Area, and Chełm Protected Landscape Area. In close proximity, there is also the Polesie National Park and the Western Polesie International Biosphere Reserve, which includes areas of the Polesie National Park. Established in 1990, the park is one of the largest protected areas in Poland, encompassing extensive wetland and forest ecosystems. This area is distinguished by its unique fauna and flora, as well as diverse soil types, making it an excellent site for studying the impact of national parks on soil ecosystems [14,15].
The proximity of such ecologically valuable areas to regions characterized by intensive human activities, such as agriculture and coal mining, indicate a strong need for monitoring the condition of these environments. Various parameters of soil microbial activity can be helpful in this regard, including biochemical activity (ammonification, nitrification), enzymatic activity (urease, protease, acid phosphatase, and fluorescein diacetate hydrolase activity), and greenhouse gas emissions, such as nitrous oxide (N2O). Ammonification and nitrification are processes responsible for the transformation of nitrogen compounds, with microbial involvement, into forms available both for the microorganisms themselves and for plants. Nitrogen is an element that is a component of proteins and nucleic acids, making its presence in the cells of living organisms essential for their proper functioning [16,17]. These processes are also closely linked to the emission of the greenhouse gas N2O, which contributes greatly to the greenhouse effect [18,19]. The intensity of ammonification and nitrification processes primarily depends on the quantity and diversity of microorganisms, as well as factors such as soil pH, temperature, moisture, and organic carbon content [20,21]. These factors also have a significant impact on the activity of protease and urease, enzymes closely linked to nitrogen transformations in soil and the soil microbiome itself [22,23,24,25]. Since nitrogen also affects the availability of phosphorus in the soil, it seems reasonable to assess the activity of acid phosphatase in this context [26,27]. On the other hand, the hydrolysis activity of fluorescein diacetate (FDA) reflects the overall microbial activity in the soil, encompassing several enzyme classes, including lipases, esterases, and proteases [28]. Biochemical and enzymatic parameters are sensitive to environmental stress and anthropogenic changes and have the potential for rapid response to these alterations [29,30,31,32].
Therefore, the activity of soil microorganisms can be used to assess not only the damage caused to the soil environment by various types of anthropogenic pressure, but also to assess the effectiveness of remedial or preventive measures, e.g., covering valuable areas with a special form of protection [15,33,34]. The cited authors point out that when assessing the condition of the soil, e.g., the effectiveness of remediation, it is important, in addition to analyzing changes in the degree of pollution, to simultaneously monitor changes in the restoration of the functionality of the soil environment. This monitoring is based on parameters related to the activity of soil microorganisms, e.g., enzymatic activity. Together with other biological and chemical properties, enzyme activities can provide useful information on the reactions of the soil to disturbances caused by pollution and support the assessment of whether and to what extent the soil has regained its health condition, and whether it is properly protected. Along with other biological and chemical properties, enzyme activities may provide useful information about the responses of soil to the disturbance caused by contamination, and support assessment of whether and to what extent the soil has regained its health conditions and whether it is properly protected. By emphasizing the relationship between enzymatic activity in soil and broader environmental processes, Daunoras et al. [34] underscore the importance of preserving soil health not only for agricultural productivity but also for ecological sustainability. In the present study, the authors used soil microbial activity parameters supporting the biogeochemical nitrogen cycle to examine and compare the state of differently used soils located in highly valuable natural areas, such as the Polesie National Park (protected area) and its surroundings. Additionally, they assessed the suitability of gangue—a waste product from the mining industry generated in the nearby “Bogdanka” coal mine—for pit reclamation and its effectiveness depending on the duration of the process (2 and 10 years). The authors also analyzed the potential of greenhouse gas N2O emissions from the aforementioned soil samples to provide a more comprehensive, although only assumed, assessment. It should be noted that the present study is part of a comprehensive project that also examined the issues discussed but is based on the activity of microorganisms responsible for the biogeochemical cycle of another main biochemical element, i.e., carbon (work in preparation). In a previous study, the authors analyzed the microbiological activity of soils in different habitats (e.g., bog, Molinia meadow, Arrhenatheretalia meadows, forest). They also assessed the effectiveness of a three-year reclamation using gangue, although the area was not subjected to cultivation [15]. In the following studies, earlier analyses were expanded to include soils from wasteland, buckwheat cultivation fields, and agricultural soils reclaimed for 2 and 10 years. Wasteland is an undeveloped area that is permanently or temporarily unused for agricultural purposes [35]. The authors formulated two research hypotheses: (i) areas subject to special forms of nature protection, such as national parks, are effectively protected from excessive human pressure exerted on the soil and air environment; (ii) the use of gangue is an effective method of restoring biological life in soils used for agriculture, although its effectiveness depends on the duration of the reclamation process. In order to examine the effectiveness of protective treatments in the PNP area, the authors compared the activities of soils (field, wasteland) located in its area with the activities of similarly used soils, but collected outside its area. In order to assess the effectiveness of reclamation treatments using gangue, the authors compared soils subjected to 2 and 10 years of reclamation (located outside the PNP) with the activity of soil from a field located outside the park because the assumption of the reclamation was that it would achieve a soil condition analogous to arable soil outside the PNP.

2. Materials and Methods

2.1. Sampling Location

Soil for analysis was collected from wastelands and cultivated fields located in the Polesie National Park (Poland, Lublin Voivodeship, 51°27′19′′ N 23°10′24′′ E), specifically: wasteland—Wielkopole (1), field—Karczunek (3), as well as areas adjacent to the park, i.e., wasteland—Zastawie (2), field—Karczunek (4) (Figure 1). Soil material was also collected from two areas reclaimed with gangue (mining waste) that differed in the duration of the reclamation process: 2 years—Wola Korybutowa Kolonia (6); 10 years—Andrzejów (5) (Figure 1). The waste was generated at the nearby Bogdanka coal mine. The reclamation involved filling the excavation site, originally agricultural land, with gangue and covering it with a 0.5 m layer of sand. During the growing season, common buckwheat (Fagopyrum esculentum Moench) was cultivated in both reclaimed areas when soil samples were collected.

2.2. Study Area Characteristics

The predominant soils in the Polesie National Park are, in order of prevalence: hydrogenic soils (peat, muck, and silt soils), which cover 48% of the park’s area; semihydrogenic soils (black soils, Gleysols, Stagnic Gleysols, and Podzolic Gleysols), accounting for 23% of the area; and autogenic soils (Brunic Regosols), which make up 19% of the park’s area [36]. More detailed information on the Polesie National Park and its surroundings can be found in the previous study [15].
Selected physical, chemical, and physicochemical properties relevant to soil microbial activity were determined in the collected soil samples to provide a more comprehensive characterization of the soils analyzed (Table 1).
The water holding capacity (WHC) of the soils collected was determined based on the difference in weight of soil samples soaked in water and then dried for 2 h [37]. Organic carbon (TOC) was determined using IR spectrometry. The Kjeldahl method was used to determine total nitrogen (TN) content.
The climatic conditions of the research area, including precipitation and temperature during the study period, are presented in Figure 2 [38].

2.3. Soil Sample Collection

Soil samples were collected from the 0–20 cm layer in 2024 during summer (June)—first sampling period, and in autumn (October)—second sampling period. The material was collected in plastic bags using an auger from four randomly selected spots within each location. Subsequently, the material from each location was averaged separately and sieved through a 2 mm mesh. The prepared soil samples were stored in plastic bags in the dark at a temperature of 4 °C.

2.4. Biochemical Analysis of N-Related Soil Processes

The intensity of the ammonification process was measured in 25 g soil samples with the addition of 0.1% asparagine. After three days of incubation, ammonium ions were extracted by mixing the samples for 20 min in 2 M KCl, followed by determining their concentration using the Nessler method [39]. The results were expressed as mg N-NH4 kg−1 d.m. of soil 3 d−1.
The intensity of the nitrification process was determined in 25 g soil samples using 0.1% ammonium phosphate as a substrate. After 7 days of incubation, nitrate ions were extracted by mixing the samples for 20 min in 2 M KCl, followed by determining their concentration using the brucine method [39]. The results were expressed as mg N-NO3 kg−1 d.m. of soil 7 d−1.
To determine N2O emission, laboratory incubations of soil samples were conducted under controlled temperature and soil moisture conditions. Soil samples (10 g, dw) were placed in 120 mL glass bottles and moistened to 60% WHC by adding distilled water. Before the main laboratory experiment, the soil samples were pre-incubated at 25 °C in the dark for three days to stabilize the soil microbial community and to avoid the impact of soil sieving [40]. After ventilating the vessels, the samples were sealed and incubated for 8 days in the dark at a temperature of 25 °C. Measurements of N2O concentration in the headspace were conducted using gas chromatography (GC). Moreover, a standard with 0.5% N2O was analyzed by GC to validate the results. The GC instrument (Perkin Elmer Clarus 500) was equipped with an elector capture detector (ECD with a temperature of 150 °C) and a flame ionization detector (FID with a temperature of 400 °C). Helium was used as the carrier gas, with a flow rate of 5 cm3 min−1.

2.5. Enzymatic Analyses

Protease activity was determined in 2 g soil samples using sodium caseinate as a substrate [41]. The samples were incubated for 1 h in 0.2 M Tris-HCl buffer at pH 8.0 and a temperature of 50 °C. The level of tyrosine released was measured spectrophotometrically, and protease activity was expressed as mg of tyrosine kg−1 d.m. of soil h−1. Urease activity was determined in 10 g soil samples using urea as a substrate [42]. The samples were incubated for 18 h at 37 °C. Ammonium ion concentration was measured spectrophotometrically, and the enzyme activity was expressed as mg NH4+ kg−1 d.m. of soil 18 h−1. The activity of acid phosphatase was determined in 1 g soil samples, to which p-nitrophenyl phosphate disodium salt (PNPNa) was added as a substrate [43]. The prepared samples were incubated for 1 h in a universal buffer at pH 6 and a temperature of 37 °C. The activity of this enzyme was measured spectrophotometrically and expressed as mg PNP kg−1 d.m. of soil h−1. Fluorescein (FDA) hydrolytic activity was determined in 1 g soil samples using fluorescein diacetate as a substrate [44]. The samples were incubated for 2 h in a 60 mM sodium phosphate buffer at pH 7.6 and a temperature of 25 °C. The enzymatic activity was measured spectrophotometrically and expressed as mg fluorescein kg−1 d.m. of soil h−1. Biochemical and enzyme activity measurements were performed using a UV-1800 spectrophotometer (Rayleigh, Beijing, China).

2.6. Statistical Analysis

All analyses were performed in triplicate. Descriptive statistics concerned calculating the arithmetic means of three replicates obtained for a given sample, along with the standard deviation. An analysis of variance (ANOVA) was performed to evaluate the statistical variability of the results, followed by Tukey’s HSD test to determine statistically significant differences. Significance was assumed at p < 0.05. Pearson’s correlation analysis was also performed at three levels of significance: p < 0.001, p < 0.01, and p < 0.05. The results are presented as a heatmap. Additionally, the relationships between soils located in and outside the Polesie National Park (PNP) and reclaimed soils were assessed based on normalized data concerning the properties of soils under study. These relationships were also presented in the form of a heatmap [45]. Statistical analysis was carried out using the Statistica 13.1 software package13.1 (TIBCO Software Inc.; Palo Alto, CA, USA).

3. Results

Table 2 and Figure 3 present the results regarding the intensity of ammonification in the soils studied. Significant differences were observed between the habitats as well as their locations (within and outside the park). The average values of this biochemical parameter (Table 2) ranged from 20.62 mg kg−1 (soil in the second year of reclamation) to 123.5 mg kg−1 (field in the park). The ammonification activity in wasteland was higher in soil sampled outside the park (51.72 mg kg−1). In contrast, the soil from cultivated fields showed higher ammonification activity in the park (123.5 mg kg−1) compared to soil outside the park (51.86 mg kg−1). The analysis of reclaimed soils showed that a longer reclamation period (10 years) led to higher activity of this process, reaching 31.97 mg kg−1, while a 2-year reclamation period resulted in the activity of only 20.62 mg kg−1. In both reclaimed soils, ammonification activity did not reach the intended level, i.e., comparable to that observed in cultivated soil outside the park. The results at individual time points showed a higher intensity of the ammonification process in autumn (Figure 3).
The results concerning nitrifier activity are presented in Table 2 and Figure 4. Significant differences in nitrification activity were also observed between soils from different habitats and locations. The average values ranged from 49.44 mg kg−1 (field soil in the park) to 12.28 mg kg−1 (soil subjected to 10 years of reclamation) (Table 2). The nitrification process, similar to ammonification, was more intense in soil from the field located in the park (49.44 mg kg−1) compared to that outside the park (16.86 mg kg−1). The soils of wasteland exhibited similar nitrifier activity regardless of location (44.35 mg kg−1 and 46.68 mg kg−1). In reclaimed soils, the nitrification process showed greater intensity in those subjected to shorter reclamation periods (37.46 mg kg−1) compared to soils undergoing long-term reclamation (12.28 mg kg−1), contrary to the trends observed for ammonification. The nitrification process in soils subjected to shorter reclamation times reached a significantly higher level (37.46 mg kg−1) compared to the target value observed in cultivated field soil outside the park (16.86 mg kg−1). The analysis of seasonal variations revealed certain differences in the intensity of nitrification across individual time points (Figure 4). These differences were most pronounced in reclaimed soils, where nitrification activity varied depending on the duration of reclamation, and was higher in summer (2-year reclamation) and in autumn (10-year reclamation).
Laboratory incubations of the samples showed that the soils tested were a source of N2O, with emission rates ranging from 0.411 ± 0.013 mg N kg−1 to 0.697 ± 0.207 mg N kg−1 in summer, and from 0.521 ± 0.016 mg N kg−1 to 0.676 ± 0.088 mg N kg−1 in autumn (Figure 5). There were greater differences between the soils collected in summer than in autumn, and in soil subjected to 10 years of reclamation (R10) emitted the most N2O in summer. The average gas emission values within a given habitat revealed that soil from the field located in the park had significantly lower emissions (0.047 mg N kg−1) compared to soil from the field outside the park (0.054 mg N kg−1) (Table 2). No other significant differences were observed within the remaining habitats or between them (Table 2).
Table 2 and Figure 6 present the results regarding urease activity. Significant differences were observed in soils depending on their use and location. The average values of this enzymatic parameter ranged from 269.45 mg kg−1 (wasteland outside the park) to 29.18 mg kg−1 and 19.46 mg kg−1 (2-year reclaimed soil and wasteland in the park) (Table 2). Cultivated soils exhibited higher urease activity outside the park (74.08 mg kg−1) compared to soils within the park (65.24 mg kg−1). A pronounced difference was observed in reclaimed soils, where significantly higher activity was recorded in soil subjected to a 10-year reclamation (72.41 mg kg−1). Thus, it reached the target level, equivalent to that observed in the soil from fields outside the park (72.08 mg kg−1). Seasonal changes presented in Figure 6 revealed that this parameter reached significantly higher values in autumn.
The results for the next enzyme involved in microbiological nitrogen transformations, i.e., protease, are presented in Table 2 and Figure 7. Statistical analysis for this enzyme also revealed significant differences in the soils of individual habitats and their locations. However, these differences were less pronounced compared to the other parameters. The average values of this parameter (Table 2) ranged from 12.65 to 12.98 mg kg−1 in the wasteland soil outside the park and the soil reclaimed for 10 years, to 1.09–1.37 mg kg−1 in the wasteland soil in the park and the soil reclaimed for 2 years. An analysis of protease activity in wastelands revealed its higher activity outside the park (12.65 mg kg−1). Similarly, cultivated soils exhibited higher proteolytic activity outside the park (2.90 mg kg−1) compared to within the park (2.06 mg kg−1). A comparison of protease activity in reclaimed soils revealed that soil subjected to longer reclamation exhibited higher activity (12.98 mg kg−1) than soil in its second year of reclamation (1.37 mg kg−1). Statistical analysis showed that the two-year reclamation resulted in an activity level comparable to that of cultivated soil in the park. Protease activity presented for different seasons (Figure 7) showed that it was generally higher in autumn.
Acid phosphatase was the following enzyme analyzed in this study. This enzyme catalyzes the transformation of phosphorus compounds, whose availability is regulated by soil nitrogen. The data obtained on phosphatase activity are presented in Table 2 and Figure 8. The data showed that, similar to protease and urease, the activity of acid phosphatase also varied between the different soils of the PNP and its surroundings, as well as in the reclaimed soils. The mean activity of this enzyme ranged from 37.04 to 38.17 mg kg−1 in cultivated field soils to 7.92 mg kg−1 in the soil reclaimed for 2 years (Table 2). A significant difference in phosphatase activity was observed between the cultivated field in the park, which exhibited lower activity (37.04 mg kg−1), and the field located outside the park (38.04 mg kg−1). A similar trend was observed for wastelands, where the soil collected in the park also showed lower phosphatase activity (12.14 mg kg−1) compared to the soil from outside the park (34.95 mg kg−1). Long-term reclamation, similar to the previous enzymes, also resulted in higher phosphatase activity (15.99 mg kg−1) compared to short-term reclamation (7.92 mg kg−1). However, even a 10-year reclamation period did not result in achieving the target activity level, i.e., comparable to that observed in soil from a field outside the park (38.17 mg kg−1). The values of acid phosphatase activity obtained at various time points indicated that this parameter generally reached higher levels in autumn, like the trends observed for the other enzymes (Figure 8).
Fluorescein hydrolase activity, similarly to the previously discussed enzymes, demonstrated significant variations depending on the habitat, location, and duration of reclamation (Table 2 and Figure 9). The average value of this parameter ranged from 120.78 mg kg−1 (field in the park) to 9.98 mg kg−1 (soil in the second year of reclamation). The soil from wasteland in the park had significantly lower hydrolytic activity (57.99 mg kg−1) compared to the soil from outside the park (87.44 mg kg−1). An opposite trend was observed for cultivated soils, where the soil in the park showed higher hydrolytic activity (120.78 mg kg−1) compared to the soil outside the park (80.90 mg kg−1). A comparison of hydrolytic activity in reclaimed soils revealed that it was higher in soil subjected to 10 years of reclamation (56.05 mg kg−1) than in soil in its second year of reclamation (9.98 mg kg−1). It should be noted that, as with most of the previously discussed parameters, long-term reclamation proved more effective but still insufficient to achieve the level of hydrolytic activity observed in cultivated soil outside the park (80.90 mg kg−1). Seasonal variations across different habitats and locations (Figure 9) did not reveal a consistent trend; for some sites, the parameter values were higher in autumn, while for others in summer.

4. Discussion

Homeostasis of the soil environment is the result of a number of adaptive mechanisms used by soil microorganisms in response to changes, e.g., related to the availability of nutrient substrates or pollution [46]. The results generally indicated lower activity of the parameters analyzed in soils from the park compared to those located outside the park (Figure 10).
This suggests that the protective role of the national park helps maintain environmental homeostasis. This is likely associated with the restrictions imposed by this form of nature conservation on residents and land users in these areas. The Act prohibits, among other activities, the use of chemical and biological plant protection agents and fertilizers, contamination of the area, soil degradation, or alteration of land use and purpose, changes in water conditions, and earthworks that would permanently deform the terrain’s topography [47]. Restrictions on soil fertilization within the park have resulted in lower carbon content in these soils (Table 1), which likely contributed to the reduced activity of protease, urease, and phosphatase. However, correlation analysis revealed a significant relationship with the content of this nutrient only for protease (Figure 11). The enzymes under study showed positive correlations with nitrogen content and WHC, while negative correlations with atmospheric factors (Figure 11). Numerous studies on soil enzymatics have demonstrated that enzymatic activity depends on these parameters [33,48,49,50]. Noteworthy are the strong positive correlations of acid phosphatase activity with processes related to NH4+ formation, such as ammonification and urease activity, as well as with nitrogen content (Figure 11). These observations confirm the relevance of combining studies on nitrogen processes with research on the microbial transformation of phosphorus compounds. Soil nitrogen significantly affects the availability of phosphorus (P), one of the most critical elements for plant nutrition. Campdelacreu Rocabruna et al. [51] reported that soil nitrogen regulated AcP through its positive effect on soil microbial biomass.
The soil from the field in the park showed notably high hydrolytic activity, which was the only case where these processes were more intense in the field at this location. This may have been related to the ban on pesticide application in the area, assuming that this parameter is sensitive to such chemical agents. The available literature indicates that pesticides have a negative effect on enzyme activity under certain conditions [52].
It should also be emphasized that findings on ammonification, nitrification, and N2O gas emissions further support the protective role of the park in relation to soil and air environments, although this was only assessed in the laboratory on samples from selected seasons. Higher activity of ammonifiers and nitrifiers was observed in the soil from a field located in the PNP; however, this was accompanied by lower N2O emissions compared to the soil from a field collected outside the park. Thus, the increased activity associated with the production of ammonium and nitrate forms of nitrogen does not result in increased N2O emissions into the atmosphere, i.e., there is no nitrogen loss from the soil. These observations are further confirmed by the heatmap (Figure 11), which highlights a negative correlation between the nitrification process and the emission of the gas discussed. However, this requires further analysis, especially in the field of denitrification, which is also a significant source of N2O [53]. Soil microorganisms participating in the nitrogen cycle through ammonification, nitrification, and denitrification processes produce the aforementioned nitrogen forms [54]. These observations suggest that in this habitat (cultivated field in PNP), the biogeochemical nitrogen cycle proceeds without disruptions. The bioavailable nitrogen forms (NH4+ and NO3−) generated with the involvement of these microorganisms can rather be incorporated into the biomass of cultivated plants and the microorganisms themselves, rather than being excessively emitted into the atmosphere, although this requires further field research. However, these statements require further research in the following years and must take into account, among other things, the activity of denitrifiers.
Anthropogenic N sources, such as fertilization of agricultural soils, affect soil conditions by, e.g., increasing the availability of N to microorganisms, thereby influencing N transformations in the soil. Soil from cultivated field in the park had the highest ammonification, regardless of the sampling term (Figure 3). At the same time, this soil was distinguished by low pH (Table 1), which is an important factor regulating the N mineralization. The negative correlation between soil pH and ammonification was confirmed by both our study (Figure 11) and previous works (summarized by Breugem et al. [55]). It has been reported that soil pH influenced microbial community shape and different bacterial and archaeal ammonia oxidizer phylotypes were present in soils of different pH [56]. Our results are in line with other studies of soils in different ecosystems, where both AM and FDA were also found to be highest in arable soils, and were also positively correlated with each other [57]. Moreover, our results also showed a positive and significant relationship of AM with AcP, which was partly confirmed in the cited study. High ammonification in soil from the field is an indication of the health of the soil and its capacity to supply N to plants, since its product is the NH4+ available to plants.
It is known that the land use and practices applied regulate soil N2O emissions, and the agricultural sector is the dominant source of this gas, particularly due to the nitrogen fertilization of soils [58]. This may explain the highest N2O emission in arable soil in autumn from outside the PNP in the present study. In contrast, the lowest N2O emission was observed in soil collected inside the PNP, due to the limitations of the cultivation treatments valid in the protected area of the park. However, it should be noted that both the wasteland and the reclamation emitted similar amounts of N2O regardless of location. The soils studied differed in the properties that determine the processes responsible for N transformation. In our study, N2O emission was positively correlated with soil pH and organic C content (Figure 11), which, beside nutrient and O2 availability, are key factors controlling nitrification and denitrification rates [59,60,61]. However, it should be considered that our research was only conducted under lab conditions and on soil samples taken at specific dates, which is a limitation of this work. Cyclic and long-term field studies would have provided a more accurate assessment of the soils studied as a source of N2O.
Analyzing the activities of the studied parameters in soils reclaimed with gangue, notable differences were observed between the soils depending on the duration of the reclamation process (2 and 10 years). Soil subjected to longer reclamation (10 years) was generally characterized by higher values. However, it should be noted that as these soils are located in agricultural areas, the aim of reclamation was to achieve activity levels comparable to those of soil from fields outside the park. Meanwhile, the microbiological activities studied in these soils remained significantly lower even after 10 years of reclamation. These observations suggest that longer reclamation was somewhat more effective than the 2-year process but still insufficient. Studies by other authors have shown that soil properties such as bulk density and aggregate structure changed after mixing soil with gangue [62]. However, the nature and intensity of this effect depended on the proportion and size of gangue particles. Also, under the conditions of this experiment, i.e., mixing sand with gangue, the above-mentioned properties that affect microbiological activity could improve with time. A review of the literature has indicated that soil enzyme activities are useful indicators for diagnosing soil degradation levels and monitoring the effectiveness of remediation efforts [33]. Enzyme activity depends on many factors, including chemical pollutants. Daunoras et al. [34] proved that a number of other factors, such as soil temperature, humidity, water content, pH, substrate availability, and average annual temperature and precipitation, also have a significant impact on enzyme activity. In addition, climate change has an ambiguous effect on these types of activity, causing both weakening and strengthening of the catalytic functions of enzymes. This is also confirmed by the correlations obtained in this study (Figure 11). Studies by other authors on long-term reclamation demonstrate that the positive effects of waste-based reclamation are already visible within the first few years and persist for up to 8 years after the treatment, indicating the restoration of biological activity even in the early phases of reclamation [30,31,63]. Studies on soil reclamation using waste have demonstrated that the type of waste material is a crucial factor. The most effective and long-lasting approach for restoring biological activity in reclaimed soil involves using organic waste (e.g., sewage sludge, compost), either alone or in combination with mineral materials [30,64,65,66]. However, when using organic waste such as sewage sludge, it should be taken into account that in addition to the positive aspects, i.e., high content of organic matter, N, P may contain chemical pollutants, which together with pollutants from gangue may exceed the permissible level [67]. Castagnoli et al. 2022 [68] point out that when using sewage sludge containing heavy metals, the penetration of these pollutants into the environment should be taken into account. Therefore, they recommend caution and to carry out leaching tests. Currently, there are advanced technologies for removing heavy metals from soils, but they generate high costs [69]. It also seems reasonable to cultivate legumes on the reclaimed soil for agricultural purposes, which would enable the development of plant growth-promoting rhizobacteria (PGPR). As is known, their effect is very important from the point of view of plant nutrition and their resistance to stress. This is related to the ability of these bacteria to bind nitrogen and dissolve phosphorus and potassium [70]. The improvement in microbial activity observed over time in the soils reclaimed with gangue may have been related to the release of heavy metals from this waste, especially in the initial period. It is widely known that this waste contains certain amounts of these elements, which can leach into the environment and negatively impact living organisms [71]. The sensitivity of soil microorganisms to heavy metals has been reported, among others, by Lee et al. [33] and Narendrula-Kotha and Nkongolo [72]. At the same time, the protective role of organic matter is a well-known phenomenon, as it shields microbial enzymes from contaminants [73]. The data in Table 1 show that lower nitrogen content and lower water capacity were noted in the reclaimed soils. Since these are important parameters for soil microorganisms [33], it seems that this could also be the reason for the ineffective reclaim. The above observations suggest that it is advisable to combine gangue with organic waste during reclamation, as it would likely accelerate the desired effects. Furthermore, the organic matter in the waste would not only serve as an additional nutrient source for microorganisms but also provide a protective role for enzymes against potential heavy metals released from the gangue.
Significant changes in all analyzed parameters of soil microbial activity indicate that they are useful in monitoring the condition of soils managed in various ways (protected, reclaimed, agriculturally used). Their combined use (biochemical, enzymatic parameters) allows for a comprehensive assessment of the correct course of the biogeochemical N cycle maintained by soil microorganisms.

5. Conclusions

The lower enzyme activity levels, generally observed in soils located in the park, suggest that the designation of these areas as a national park contributes to maintaining their homeostasis. Although higher activities of ammonification and nitrifiers were recorded in the cultivated soil in the park, it should be noted that this soil in autumn also generated lowest N2O emissions into the atmosphere compared to, e.g., cultivated fields outside the park. Nevertheless, this observation requires confirmation through longer-term field analyses. These findings suggest that the biogeochemical nitrogen cycle is not disrupted in the cultivated habitat within the park. The bioavailable nitrogen forms produced by microorganisms, such as NH4+ and NO3−, are incorporated into plant and microbial biomass rather than being emitted into the atmosphere as gaseous compounds. Thus, nitrogen losses from the soil do not occur through this pathway.
The assessment of the effectiveness of reclamation treatments using gangue, based on monitoring the activity of microorganisms involved in the nitrogen biogeochemical cycle, demonstrated that such reclamation is not effective. However, a longer duration of reclamation (10 years) proved more favorable than a shorter one (2 years), yet it remained insufficient, as the average activity levels measured throughout the study period were still significantly lower compared to cultivated soils. The higher activity of certain parameters (including potential of N2O emissions) observed during specific periods in this soil may indicate instability in the habitat, even after such a prolonged period, highlighting the need for continued research in subsequent years. Thus, the use of gangue alone for reclamation of excavations intended for agricultural purposes proved ineffective. Therefore, utilizing gangue for land reclamation appears reasonable, but only when combined with organic waste (e.g., sewage sludge)—an approach that requires further research, which will continue to be explored. Alternatively, a different reclamation purpose can be considered, such as recreational rather than agricultural use.
In order to increase the effectiveness of soil remediation with gangue, it seems reasonable to undertake activities aimed at enriching it with nitrogen. Good agricultural practice in this area may be, for example, growing legumes on such remediated soil or using organic fertilizers, e.g., based on waste, such as composts, mushroom waste or manure. These activities are in line with the principles of sustainable development, taking into account corrective actions, including closed-loop waste management.

Author Contributions

Conceptualization, J.J. and E.K.; methodology, J.J. and A.W.; software, E.K.; validation, J.J., E.K., and A.W.; formal analysis, J.J. and E.K.; investigation, J.J.; resources, J.J. and G.G.; data curation, E.K.; writing—original draft preparation, J.J, E.K., A.W., and G.G.; writing—review and editing, J.J. and E.K.; visualization, E.K.; supervision, J.J.; project administration, J.J.; funding acquisition, J.J. All authors have read and agreed to the published version of the manuscript.

Funding

The presented research was financed within the framework of the NB 0701-2/2024/2 (UBAD.WRM.24.104) project entitled “Parameters of microorganism activity and biodiversity and phytotoxicity as a tool for monitoring soils of the Poleski National Park”, implemented within the framework of the Strategic Program: TOGETHER FOR BIODIVERSITY from the resources of the Research and Action Fund for Environmental Protection in the Lublin Coal Basin: GRANTS FOR SCIENTISTS”, intended to finance scientific research or scientific research-implementation projects for nature and environmental protection in the PNP area, including in the aspect of direct and indirect impact of LW Bogdanka on PNP ecosystems. Additionally, part of the research was financed by the University of Life Sciences in Lublin (SUBB.WRM.19.028.RiO).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Additional information is provided by the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of sampling sites. Wielkopole (1); Zastawie (2); Karczunek (3)—in the PNP); Karczunek (4)—outside the PNP; Andrzejów (5); Wola Korybutowa Kolonia (6).
Figure 1. Distribution of sampling sites. Wielkopole (1); Zastawie (2); Karczunek (3)—in the PNP); Karczunek (4)—outside the PNP; Andrzejów (5); Wola Korybutowa Kolonia (6).
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Figure 2. Mean monthly temperatures and rainfall in the experimental site during the period of the experiment. The figure was created based on data taken from the Tutiempo website [38].
Figure 2. Mean monthly temperatures and rainfall in the experimental site during the period of the experiment. The figure was created based on data taken from the Tutiempo website [38].
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Figure 3. Ammonification in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 3. Ammonification in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 4. Nitrification in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 4. Nitrification in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 5. Cumulative soil N2O emissions. W—wasteland, CF—cultivated field, R2-reclaimed land 2 years, R10-reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 5. Cumulative soil N2O emissions. W—wasteland, CF—cultivated field, R2-reclaimed land 2 years, R10-reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 6. Activity of urease in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 6. Activity of urease in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 7. Activity of protease in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 7. Activity of protease in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 8. Acid phosphatase activity in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 8. Acid phosphatase activity in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 9. FDA hydrolytic activity in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
Figure 9. FDA hydrolytic activity in the analyzed soil. W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters above the columns indicate significant differences at p < 0.05.
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Figure 10. Heatmap based on normalized soil quality indicator data in the area inside and outside the PNP and reclaimed soils. Abbreviations: W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years, AM—ammonification, NIT—nitrification, N2O—nitrous oxide, URE—urease, PRO—protease, AcP—acid phosphatase, FDA—fluorescein diacetate hydrolysis activity.
Figure 10. Heatmap based on normalized soil quality indicator data in the area inside and outside the PNP and reclaimed soils. Abbreviations: W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years, AM—ammonification, NIT—nitrification, N2O—nitrous oxide, URE—urease, PRO—protease, AcP—acid phosphatase, FDA—fluorescein diacetate hydrolysis activity.
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Figure 11. Heatmap displaying the Pearson’s correlation coefficients between the soil’s physico-chemical, chemical, and environmental factors, and microbial properties. AM—ammonification, NIT—nitrification, URE—urease, PRO—protease, AcP—acid phosphatase, FDA—fluorescein diacetate hydrolysis activity, N2O—nitrous oxide, TN—total nitrogen, TOC—total organic carbon, RAIN—rainfall, TEMP—temperature, WHC—water holding capacity. Significant at * p < 0.05; ** p < 0.01; *** p <0.001, respectively.
Figure 11. Heatmap displaying the Pearson’s correlation coefficients between the soil’s physico-chemical, chemical, and environmental factors, and microbial properties. AM—ammonification, NIT—nitrification, URE—urease, PRO—protease, AcP—acid phosphatase, FDA—fluorescein diacetate hydrolysis activity, N2O—nitrous oxide, TN—total nitrogen, TOC—total organic carbon, RAIN—rainfall, TEMP—temperature, WHC—water holding capacity. Significant at * p < 0.05; ** p < 0.01; *** p <0.001, respectively.
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Table 1. Selected properties of the soil.
Table 1. Selected properties of the soil.
TermWastelandCultivated FieldReclaimed Land
2 Years		Reclaimed Land
10 Years
AreaAreaArea
Outside
the PNP		Area
Outside
the PNP
inOutsideinOutside
the PNPthe PNP
pH
1 mol KCl		summer5.046.184.274.147.158.25
autumn3.957.334.155.947.168.20
TOC
g kg−1		summer0.200.607.119.634.3913.74
autumn0.201.606.8212.22.4725.87
TN
g kg−1		summer4.77.40.80.70.10.6
autumn3.920.40.70.40.30.8
WHC
g H2O g dry soil−1		 0.340.500.330.430.240.33
Abbreviations: TOC—total organic carbon, TN—total nitrogen, WHC—water holding capacity.
Table 2. Biochemical and enzymatic activity in soil (averages for the entire study period).
Table 2. Biochemical and enzymatic activity in soil (averages for the entire study period).
HabitatLocationAMNITN2OUREPROAcPFDA
Warea in the PNPa 42.11 Da 44.35 Aa 0.50 Aa 19.46 Ba 1.09 Aa 12.14 Ca 57.99 A
area outside the PNPb 51.72 Aa 46.68 ABa 0.52 Ab 269.45 Cb 12.65 Cb 34.95 Eb 87.44 B
CFarea in the PNPb 123.50 Eb 49.44 Ba 0.47 Aa 65.24 Aa 2.06 ABa 37.04 Ab 120.78 D
area outside the PNPa 51.86 Aa 16.86 Db 0.54 Ab 74.08 Ab 2.90 Bb 38.17 Aa 80.90 B
R2area outside the PNPa 20.62 Bb 37.46 Ea 0.55 Aa 29.18 Ba 1.37 Aa 7.92 Ba 9.98 C
R10b 31.97 Ca 12.28 Ca 0.66 Ab 72.41 Ab 12.98 Cb 15.99 Db 56.05 A
Abbreviations: W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years, AM—ammonification (mg N-NH4 kg−1 d.m. of soil 3 d−1), NIT—nitrification (mg N-NO3 kg−1 d.m. of soil 7 d−1), N2O—nitrous oxide (mg N kg−1), URE—urease (mg NH4 kg−1 d.m. of soil 18 h−1), PRO—protease (mg tyrosine kg−1 d.m. of soil 18 h−1), AcP—acid phosphatase (mg PNP kg−1 d.m. of soil h−1), FDA—FDA hydrolytic activity (mg fluorescein kg−1 d.m. of soil h−1). Different letters indicate significant differences, according to Tukey’s test, at p < 0.05. Lower case letters denote homogeneous groups defined separately for each habitat and a given parameter. Upper case letters denote homogeneous groups defined jointly for all habitats for a given parameter.
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MDPI and ACS Style

Joniec, J.; Kwiatkowska, E.; Walkiewicz, A.; Grzywaczewski, G. Changes in Microbial Activity Associated with the Nitrogen Biogeochemical Cycle in Differently Managed Soils, Including Protected Areas and Those Reclaimed with Gangue. Sustainability 2025, 17, 4343. https://doi.org/10.3390/su17104343

AMA Style

Joniec J, Kwiatkowska E, Walkiewicz A, Grzywaczewski G. Changes in Microbial Activity Associated with the Nitrogen Biogeochemical Cycle in Differently Managed Soils, Including Protected Areas and Those Reclaimed with Gangue. Sustainability. 2025; 17(10):4343. https://doi.org/10.3390/su17104343

Chicago/Turabian Style

Joniec, Jolanta, Edyta Kwiatkowska, Anna Walkiewicz, and Grzegorz Grzywaczewski. 2025. "Changes in Microbial Activity Associated with the Nitrogen Biogeochemical Cycle in Differently Managed Soils, Including Protected Areas and Those Reclaimed with Gangue" Sustainability 17, no. 10: 4343. https://doi.org/10.3390/su17104343

APA Style

Joniec, J., Kwiatkowska, E., Walkiewicz, A., & Grzywaczewski, G. (2025). Changes in Microbial Activity Associated with the Nitrogen Biogeochemical Cycle in Differently Managed Soils, Including Protected Areas and Those Reclaimed with Gangue. Sustainability, 17(10), 4343. https://doi.org/10.3390/su17104343

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