Biogeochemical Carbon Cycling and Phytotoxicity of Soils Under Various Land-Use Types, Including Those Reclaimed Using Gangue

Abstract

Land use strongly influences carbon (C) cycling and its sequestration in soils, which is a key component of sustainable development due to its role in ecosystem stability. The contribution of microorganisms to the biogeochemical C cycle was studied in soils located within the protected area of Polesie National Park (PNP) and in similarly man-aged soils outside the park. These phenomena were also assessed in degraded soils subjected to 2- and 10-year reclamation using gangue. The results showed significant differences depending on the location, particularly in fallow lands. In these areas, bacterial counts, DNA concentration, respiratory activity, and enzymatic activity were lower in soils within the park, while fungal populations exhibited more intensive growth. Park soils also demonstrated more favorable phytotoxicity indicators, including seed germination (wasteland—92 and cultivated field—83 seeds) and root growth (cultivated field—2.3 cm). Reclamation proved ineffective, especially within the first two years. Studies indicated that reclamation based solely on gangue is insufficient, not entirely safe, and requires a longer timeframe. From the perspective of sustainable development, this highlights the need for reclamation methods that ensure responsible use of waste materials, support long-term soil quality improvement, and protect ecosystem integrity. The results emphasize the importance of protective measures in valuable natural areas and inform decisions regarding the use of gangue in reclamation.

1. Introduction

Carbon (C), alongside nitrogen (N), is one of the most important and indispensable elements for the proper functioning of living organisms and entire ecosystems. It shows a particular ability to bond with other elements, such as nitrogen, oxygen, phosphorus, and hydrogen, to form organic molecules and mineral compounds. This element is a fundamental component of the carbon cycle, responsible for maintaining its balance between living organisms, soil, oceans, and the atmosphere [1]. This process contributes to plant growth through photosynthesis, C sequestration in natural sinks, and the regulation of ecosystem functions, all of which influence the regulation of Earth’s climate [1,2]. Microorganisms, particularly the diverse community of soil microorganisms, play a key role in all these stages. This is due to the fact that, on a global scale, soil stores more carbon than the atmosphere and vegetation combined [3].

The soil microbiome is the most abundant and diverse community in the biosphere, and among its many functions in soil, it is responsible for the decomposition of organic matter, C storage, and nutrient cycling [2,4]. Heterotrophic microorganisms play a particularly important role in this context, as their unique metabolic functions and diversity increasingly make them a key focus in research on biological C sequestration and the efficient use of these resources [5,6]. Heterotrophic microorganisms utilize carbon stored in residues of other microorganisms, plants, or animals as a metabolic substrate, retaining part of the carbon in their biomass while releasing the remainder as metabolites or carbon dioxide (CO₂) into the atmosphere, thereby becoming a main source of this greenhouse gas (GHG) [3,7,8]. Kopittke et al. [3] identified soils as a major global source of CO₂ emissions, primarily resulting from historical losses of soil organic C due to land-use change, agriculture, and degradation. The authors emphasized that while much of this CO₂ has already been released in the past, preventing further C loss and rebuilding soil C stocks are critical strategies for climate change mitigation, because soils can also act as significant C sinks. The information presented shows that soil microorganisms are key elements of the carbon cycle and, consequently, of the Earth’s climate, but in parallel, they are highly sensitive to environmental changes [9,10,11,12,13,14]. Therefore, monitoring the soil microbiome, and thus soil quality, is crucial for a more accurate understanding of the impact of land use on soil ecosystems as well as on global climate change.

Soil enzymes are also a fundamental part of the carbon cycle and are closely associated with soil microorganisms, as, in addition to root exudates and decomposed plant and animal residues, they are a major source of enzymes in the soil environment [15,16]. Consequently, enzymes are also integral components of ecosystem processes, acting as natural catalysts in many of them, including organic matter decomposition and nutrient cycling [15]. β-glucosidase and dehydrogenases are among the most important enzymes in the soil environment. β-glucosidase ensures the availability of carbon sources for microorganisms by catalyzing the hydrolysis of glucose derivatives, thereby affecting cellulose degradation and the carbon cycle [16]. Dehydrogenases, on the other hand, represent a key stage in the biological oxidation of soil organic matter, as they are involved in transferring hydrogen from organic substrates to inorganic acceptors [16]. These enzymes are also highly sensitive to environmental changes, and thus they play a key role in soil health and fertility [15,17,18,19,20]. Consequently, understanding the mechanisms and interactions of these enzymes is a priority for improving soil productivity and promoting sustainable development in many ecosystems.

Soil respiration is another important component of the carbon cycle, driven by the activity of the soil microbiome and organic matter decomposition. Understanding processes involved in soil respiration can play a leading role in mitigating climate change, as it is responsible for releasing one of the main greenhouse gases into the atmosphere, i.e., CO₂ [3]. Soil respiration is based on two pathways: the autotrophic pathway, related to plant root activity and associated biota, and the heterotrophic pathway, which involves organic carbon decomposition by soil organisms [21]. Therefore, changes in the activity of respiratory processes may indicate ecological disturbances and also a large contribution of microorganisms to soil metabolism and global warming [13,22]. Respiratory processes, defined as the rate of CO₂ production, are a useful measure of soil microbial activity and can be applied to assess the intensity or reduction in GHG emissions, evaluate the effectiveness of reclamation, and the degree of soil degradation [13,23,24].

Undoubtedly, the activity of various groups of microorganisms contributes to the emission of key GHG, such as CO₂, methane (CH₄), and nitrous oxide (N₂O), but human activities, particularly in agriculture and waste management, also significantly influence these emissions by stimulating microbial processes [22,25,26]. Mining, which is an important part of industrial development, and thus plays a key role in the economy, not only poses a significant risk of heavy metal pollution [27] but is also a notable emitter of GHG [28]. According to Zheng et al. [28], GHG emissions from the mining industry reached 7.7 billion tons of CO₂ equivalents (CO₂e) in 2020, accounting for approximately 15.0% of global anthropogenic GHG emissions. Therefore, monitoring should be prioritized not only in areas directly affected by mining activities but also in neighboring regions [29]. Moreover, mining is also associated with the generation of large amounts of various types of waste, including gangue [30,31]. Despite its drawbacks, such as contribution to soil erosion and depleting nutrients, organic matter, and other components [32,33], this waste is increasingly being utilized in industry and reclamation [32,34,35]. Therefore, a key aspect of efforts to utilize this waste should be the assessment of its impact on the soil environment, including its effects on microorganisms, which are its primary driving force.

Land use also plays a crucial role in the proper functioning of the soil environment. It affects the physical and chemical properties of soil, which in turn alters the microhabitats in which soil microorganisms are present, ultimately influencing carbon cycling in ecosystems and contributing to global climate change [25,36,37].

Therefore, the present study employed various microbiological parameters related to carbon cycling, including CO₂ emissions as a GHG, and phytotoxicity indicators, to assess and compare soil condition under different land uses. The current study assessed arable fields and fallow lands located both within and outside the Polesie National Park (PNP), a highly valuable and unique area in terms of natural heritage, protected under a special form of nature conservation [29]. Furthermore, the experiments evaluated the effectiveness of 2- and 10-year reclamation using gangue from the Bogdanka coal mine, located in close proximity to the PNP. The assessment was conducted by comparing the activity of reclaimed soils with soil from a field outside the park, which represented the target condition of the reclamation. The novelty of this work is also reflected in the unique proximity (approximately 10 km) of such diverse areas as a coal mine, a national park, and intensively farmed land. This study is part of a broader research project in which previous investigations in the same study area focused primarily on microbial processes associated with the nitrogen cycle [29,38]. However, microbial processes related to the carbon cycle and their relationship with phytotoxicity have not yet been assessed within this comparative framework. This represents an important research gap because carbon- and nitrogen-related microbial processes provide complementary information on soil functioning and may respond differently to land use and reclamation.

The present study addresses this gap by integrating multiple indicators of microbial functioning related to the carbon cycle, including bacterial and fungal abundance, DNA concentration, enzymatic activity, and soil respiration assessed through CO₂ emissions, with phytotoxicity indicators based on seed germination and root elongation of Lepidium sativum L. These parameters were assessed within the same experimental framework across protected and non-protected soils under different land uses and in areas reclaimed with coal-mining gangue at two reclamation ages. This integrated approach extends our previous assessment based on nitrogen-cycle processes and provides complementary information on microbial functioning and phytotoxicity in soils subjected to different land-use and reclamation conditions. This approach aligns with the current use of microbiological indicators as sensitive bioindicators of soil condition and health, including in studies on ecosystem restoration and the sustainable management of soil resources [39]. The following hypotheses were proposed: (i) the national park status provides an effective and sufficient form of protection that supports soil homeostasis in terms of microbial C cycling and phytotoxicity; (ii) land-use types (field, fallow land, reclaimed area) do not contribute to excessive GHG emissions; (iii) the use of gangue (a waste product from the coal mining industry) is an effective method for soil reclamation, promoting the restoration of biological activity without increasing phytotoxicity.

2. Materials and Methods

2.1. Description of the Research Area

Soil for analysis was collected from fallow lands and agricultural fields and within the Polesie National Park (PNP; Poland, Lublin Voivodeship, 51°27′19″ N 23°10′24″ E), specifically: fallow land–Wielkopole (1), field–Karczunek (3), as well as areas adjacent to the park, i.e., fallow land–Zastawie (2), field–Karczunek (4) (Figure 1). Moreover, the study included soils from areas reclaimed with gangue for 2 years at Wola Korybutowa Kolonia (6) and for 10 years at Andrzejów (5) (Figure 1). The waste material used for reclamation originated from the nearby Bogdanka hard coal mine. For land reclamation, the excavation was backfilled with gangue and covered with a 0.5 m layer of sand. During the growing season analyzed in the study, common buckwheat (Fagopyrum esculentum Moench) was cultivated at both reclaimed sites.

Figure 1. Distribution of sampling sites. In the PNP: 1—Wielkopole; 3—Karczunek; outside the PNP: 2—Zastawie, 4—Karczunek; 5—Andrzejów; 6—Wola Korybutowa Kolonia.

The predominant soils in the Polesie National Park are: hydrogenic soils (peat, muck, and mud soils), covering 48% of the park area; semihydrogenic soils (black earths, groundwater gley soils, stagnic gley soils, and gley–podzolic soils), accounting for 23% of the park area; and autogenic soils (Brunic Regosols), representing 19% of the park area [40]. More detailed information on the PNP and its surroundings is provided in an earlier study [29].

Considering the importance of climatic conditions for soil microbial activity, Figure 2 illustrates the precipitation and temperature patterns recorded in the study area during the investigation period.

Figure 2. Mean monthly rainfall (A) and temperatures (B) in the experimental site during the period of the experiment. The figure was created based on data taken from the Tutiempo website [41].

2.2. Soil Sampling and Baseline Characteristics of Soil Samples

Soil samples were collected from the 0–20 cm layer during the summer (June)—first sampling period, and in autumn (October)—second sampling period, in 2024. The material was collected in plastic bags using an auger. Soil samples were collected following the general principles of the LUCAS soil sampling methodology [42]. Four subsamples were collected from each study site and thoroughly homogenized to obtain one composite sample representative of the site. They were subsequently sieved through a 2 mm mesh. The soil samples were kept in plastic bags, and stored in the dark at 4 °C.

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 analyzed soils (Table 1). Additionally, the content of selected heavy metals was determined in the reclaimed soils and in soil from a field located outside the park (Table 2).

Table 1. Selected properties of the soil [38].

	Term	Wasteland		Cultivated Field		Reclaimed Land 2 Years	Reclaimed Land 10 Years
		Area in Outside the PNP		Area in Outside the PNP		Area Outside the PNP	Area Outside the PNP
pH 1 mol KCl	summer	5.04	6.18	4.27	4.14	7.15	8.25
	autumn	3.95	7.33	4.15	5.94	7.16	8.20
TOC g kg−1	summer	0.20	0.60	7.11	9.63	4.39	13.74
	autumn	0.20	1.60	6.82	12.2	2.47	25.87
TN g kg−1	summer	4.7	7.4	0.8	0.7	0.1	0.6
	autumn	3.9	20.4	0.7	0.4	0.3	0.8
WHC g H2O g dry soil−1		0.34	0.50	0.33	0.43	0.24	0.33

Abbreviations: TOC—total organic carbon, TN—total nitrogen, WHC—water-holding capacity.

Table 2. Heavy metals and arsenic contents in the soil.

Heavy Metals	Unit	Cultivated Field	Reclaimed Land 2 Years	Reclaimed Land 10 Years
		Area Outside the PNP	Area Outside the PNP	Area Outside the PNP
As	mg/kg	0.30	0.46	0.93
Cd		<LOQ = 0.02	<LOQ = 0.02	<LOQ = 0.02
Pb		3.93	2.07	4.40
Hg		0.01	0.01	0.02

Abbreviations: LOQ—Limit of Quantification.

Water-holding capacity (WHC) of the collected soils was calculated from the difference in mass between samples saturated with water and subsequently dried for 2 h [43]. Total organic carbon (TOC) was measured by infrared spectrometry. Total nitrogen (TN) was determined using the Kjeldahl method.

Elemental concentrations were determined in a laboratory accredited to ISO/IEC 17025:2017 [44]. As, Cd, and Pb were determined by ICP-MS, while Hg was determined by atomic absorption spectrometry (AAS) with amalgamation using an AMA-254 analyzer (Altec, Brmo, Czech Republic).

2.3. Microbiological Analysis

Populations of individual bacterial and fungal groups in the soil samples were determined using the plate count method and the enumeration procedure published by Foght and Aislabie [45]. Analyses were performed in triplicate. Results were expressed per 1 kg of soil dry weight as colony-forming units (CFU) or as the most probable number (MPN) based on McCrady’s tables. Microbiological analyses included determination of oligotrophic bacteria on a medium containing soil extract and K₂HPO₄; filamentous fungi on Martin medium [46]; cellulolytic bacteria in liquid medium according to Pochon and Tardieux [47], with MPN values derived from McCrady’s tables; and cellulolytic fungi on mineral agar supplemented with antibiotics in the amounts recommended by Martin [46] and overlaid with a Whatman paper disc. Bacterial cultures were incubated at 28 °C for 4 days to quantify oligotrophic bacteria and for 14 days to identify and enumerate cellulolytic bacteria. Fungal cultures were incubated at 25 °C for 3 days to determine filamentous fungi and for 14 days to quantify cellulolytic fungi.

2.4. DNA Extraction

Total DNA was extracted from 100 mg of soil using the Soil DNA Purification Kit (EurX) according to the manufacturer’s instructions. The extracted DNA was used for DNA concentration assessment only. DNA concentration was measured with a NanoDrop One UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

2.5. Biochemical Activity

Substrate-induced respiration (SIR) was determined by titration through measurement of CO₂ released from glucose-amended soil [48]. Analyses were performed on 20 g samples of fresh soil supplemented with 1% glucose and incubated in flasks containing 0.2 M NaOH. After 24 h incubation at 25 °C, the excess NaOH was titrated with 0.1 M HCl in the presence of phenolphthalein. CO₂ production was expressed as mg C–CO₂ per kg soil dry matter per day.

Basal respiration (R), reflecting potential CO₂ emissions, was determined using soil samples of approximately 450 g dry weight placed in 5-dm³ airtight dark vessels (n = 3). The soils were adjusted to 60% of water-holding capacity with distilled water and preincubated for 3 days at 25 °C in the dark to equilibrate moisture conditions and stabilize indigenous microbial communities [49]. After preincubations, the vessels were ventilated and resealed. Incubation continued at 25 °C under laboratory conditions [50]. CO₂ concentration in the headspace was monitored for 6 h at approximately hourly intervals using a Gasmet DX 4040 Fourier Transform Infrared Gas Analyzer (Gasmet Technologies Oy, Helsinki, Finland). The CO₂ emission rate was calculated as the difference between final and initial CO₂ concentrations divided by the incubation time and expressed as mg C–CO₂ per kg soil dry matter per day.

2.6. Enzymatic Activity

Dehydrogenase activity was determined according to the method of Thalmann [51]. Soil samples (5 g) were incubated with 2,3,5-triphenyltetrazolium chloride as substrate in 0.1 M tris(hydroxymethyl)aminomethane buffer (Tris–HCl, pH 7.4) for 48 h at 30 °C. Enzymatic activity was measured colorimetrically at 485 nm and expressed as mg triphenyl formazan (TPF) per kg soil dry weight per day. β-glucosidase activity was determined in 1 g soil samples incubated in modified universal buffer (pH 6.0) for 1 h at 37 °C, using p nitrophenyl β D glucopyranoside as substrate [52]. Activity was measured colorimetrically at 400 nm and expressed as mg p-nitrophenol (PNP) per kg soil dry weight per hour. Enzyme activity measurements were performed using a UV-1800 spectrophotometer (Ray-leigh, Beijing, China).

2.7. Phytotoxicity Test

The assay was conducted on soil collected in spring and assessed the effects of potentially toxic substances present in the soil solution on germination and early root growth of Lepidium sativum L. after 2 days. Fresh soil samples (20 g; 60% WHC) were placed in Petri dishes lined with sterile blotting paper in six replicates. Ninety seeds were sown on three dishes and ten seeds on the remaining three. Incubation was carried out at 22 °C. After 2 days, the number of germinated seeds was recorded on all plates. Root length was measured on the plates containing ten seeds.

2.8. Statistical Analysis

Statistical analyses were carried out using Statistica 13.1 (TIBCO Software Inc.; Palo Alto, CA, USA). Differences in soil properties between land-use types (fallow, cultivated field, reclaimed area) were assessed using the Kruskal–Wallis test, and when significant differences were detected (p < 0.05), the Dunn post hoc test with Bonferroni correction was applied. Nonparametric methods were used because the assumptions of analysis of variance were not met. Additionally, based on normalized data, the relationships between soils from inside and outside the PNP and reclaimed soils were assessed and presented as a heatmap [53]. Pearson’s correlation analysis was also performed at three levels of significance (p < 0.001; p < 0.01; p < 0.05), and the results were presented in the form of a heatmap. Principal component analysis (PCA) was used to further assess the relationships between the studied parameters and to identify the main factors differentiating the soil samples.

3. Results

The abundance of oligotrophic bacteria in the soil samples is presented in Figure 3A and Table 3. This parameter showed significant differences between individual habitats (field, fallow, reclaimed soil), as well as between their locations and sampling times. The mean values ranged from 1.01 to 3.06 × 10⁹ CFU kg⁻¹ (Figure 3A). Bacterial abundances in both fallow and cultivated fields were lower in the park (1.01 and 2.42 × 10⁹ CFU) than outside it (2.45 and 2.99 × 10⁹ CFU). Seasonal analysis showed that this effect was particularly pronounced in autumn (Table 3).

Figure 3. Number of oligotrophic bacteria (A) and cellulolytic bacteria (B) in the analyzed soil (averages for the entire study period). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

Table 3. Microbiological, biochemical and enzymatic activity of the tested soils in individual seasons.

Habitat	Location	Season	OB	FF	CB	CF	DNA	SIR	R	DEH	B-GLU
W	area in the PNP	s	1.71 ac	76.77 c	0.07	22.52 ab	7.78 ab	57.27 a	2.91 abc	0.38 bcd	14.13 f
		a	0.31 d	149.28 g	0.04	36.81 ef	7.57 ab	28.53 c	4.51 b	0.17 a	8.73 b
	area outside the PNP	s	1.02 cd	25.61 a	1.04	14.27 d	18.17 d	85.99 e	2.59 ac	0.39 bd	41.29 a
		a	3.87 e	29.54 ab	0.40	24.17 bc	41.53 e	101.82 d	4.60 b	1.94 f	74.15 h
CF	area in the PNP	s	2.71 bf	116.07 de	4.57	49.75 h	22.00 d	49.28 a	2.39 a	0.50 de	38.58 ae
		a	2.13 ab	124.51 ef	0.80	45.36 gh	13.52 c	25.61 bc	3.64 abc	0.29 abc	30.43 g
	area outside the PNP	s	2.46 ab	69.08 c	0.09	40.24 fg	13.73 c	55.41 a	3.27 abc	0.59 e	36.25 de
		a	3.51 ef	100.80 d	0.99	34.76 e	21.37 d	23.85 bc	1.94 a	0.59 e	34.58 d
R2	area outside the PNP	s	0.94 cd	22.22 a	0.26	23.96 abc	7.76 ab	45.65 a	2.26 a	0.21 a	6.48 b
		a	2.25 ab	45.96 b	11.58	28.07 c	4.28 a	15.09 b	3.04 abc	0.30 abc	18.54 c
R10		s	1.80 abc	24.90 a	1.56	18.67 ad	9.90 bc	185.29 f	4.53 b	0.41 bd	21.45 c
		a	4.31 e	135.47 fg	14.97	55.97 i	13.54 c	110.27 d	4.35 bc	0.23 ac	41.21 a

Abbreviations: W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; s—summer, a—autumn; OB—oligotrophic bacteria (cfu 10⁹ kg⁻¹ d.m. of soil), FF—filamentous fungi (cfu 10⁶ kg⁻¹ d.m. of soil), CB—cellulolytic bacteria (10⁵ kg⁻¹ d.m. of soil), CF—cellulolytic fungi (cfu10⁶ kg⁻¹ d.m. of soil), DNA—dsDNA concentration (μg g⁻¹ d.m. of soil), SIR—substrate induced respiration (mg C-CO₂ kg⁻¹ d.m. of soil d⁻¹), R—respiration (mg C kg⁻¹ d.m. of soil d⁻¹), DEH—dehydrogenases (mg TPF kg⁻¹ d.m. of soil d⁻¹), B-GLU—β-glucosidase (mg PNP kg⁻¹ d.m. of soil h⁻¹); different letters indicate the significance of the differences.

A similar trend was observed for cellulolytic bacteria (Figure 3B, Table 3). For fallow soils, the mean abundance of these bacteria was also lower within the park (0.06 × 10⁶ kg⁻¹) than outside it (0.72 × 10⁶) (Figure 3B). This effect persisted during both sampling periods (Table 3). In soils collected from fields, the mean value of this parameter was higher for the field located within the park (2.69 × 10⁶) than outside it (0.54 × 10⁶). However, the data on changes at individual sampling times (Table 3) clearly confirmed this only in summer, whereas in autumn, the abundance was slightly higher in soil outside the park.

Data on the abundance of oligotrophic bacteria in reclaimed soils showed that their levels varied significantly depending on the duration of reclamation (Figure 3A, Table 3). Soil subjected to 2 years of reclamation had a lower bacterial abundance (1.59 × 10⁹ CFU kg⁻¹) than the target level, i.e., soil from a field outside the park (2.99 × 10⁹ CFU). In contrast, soil subjected to 10 years of reclamation had a significantly higher bacterial abundance than the field soil, reaching 3.06 × 10⁹ CFU. For cellulolytic bacteria (Figure 3B, Table 3), both reclaimed soils had larger populations of these microorganisms (5.92 and 8.27 × 10⁶) than the field soil outside the park (0.54 × 10⁶). Moreover, 10 years of reclamation resulted in a significantly higher development of this bacterial group compared to 2 years of reclamation. Changes at individual sampling times for oligotrophic and cellulolytic bacteria showed that their abundance was significantly higher in both reclaimed soils in autumn (Table 3).

The data presented in Figure 4 and Table 3 indicate that, similar to bacterial abundance, the population size of filamentous and cellulolytic fungi also differed significantly between habitats, locations, and sampling times. The mean values for the abundance of filamentous fungi ranged from 27.57 to 120.29 × 10⁶ CFU kg⁻¹ (Figure 4A), while those for cellulolytic fungi varied from 19.22 to 47.55 × 10⁶ CFU (Figure 4B). In contrast to bacteria, fungal development was more intensive in soils within the park (113.02 and 120.29 × 10⁶ CFU for filamentous fungi; 29.66 and 47.55 × 10⁶ CFU for cellulolytic fungi) than outside it (27.57 and 84.94 × 10⁶ CFU for filamentous fungi; 19.22 and 37.50 × 10⁶ CFU for cellulolytic fungi). Data on the mean abundances of both fungal groups in reclaimed soils (Figure 4) indicate that their development was weaker in soil subjected to 2 years of reclamation (34.09 × 10⁶ CFU for filamentous fungi and 26.01 × 10⁶ CFU for cellulolytic fungi) than in soil from the field outside the park (84.94 × 10⁶ CFU for filamentous fungi and 37.50 × 10⁶ CFU for cellulolytic fungi). Meanwhile, 10 years of reclamation increased the abundance of these microorganisms to the target level (80.18 × 10⁶ CFU for filamentous fungi and 37.32 × 10⁶ CFU for cellulolytic fungi), i.e., that recorded in soil from outside the park. Seasonal variation analysis regarding the development of these fungal groups in both reclaimed soils showed that these parameters reached higher values in autumn (Table 3).

Figure 4. Number of filamentous fungi (A) and cellulolytic fungi (B) in the analyzed soil (averages for the entire study period). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

Data concerning dsDNA concentration indicate that this parameter varied between the soils (Figure 5, Table 3). Its mean values ranged from 6.02 to 29.85 µg g⁻¹. In fallow soils, the concentration was higher in soil located outside the park (29.85 µg) than within it (7.67 µg). The mean values of this parameter in field soils from both locations were similar and amounted to 17.76 and 17.55 µg, respectively. However, seasonal variation analysis in these soils generally revealed significant differences in dsDNA concentrations between sampling times, with higher values in autumn at the site outside the park (Table 3). Analysis of mean dsDNA concentrations in soils reclaimed for 2 and 10 years showed that they were lower than in soil from the field outside the park (17.55 µg—target level). Significant differences in the level of this parameter were also observed between individual soils. Soil reclaimed for 2 years was characterized by a significantly lower dsDNA concentration (6.02 µg) than soil reclaimed for 10 years (11.72 µg). Seasonal analysis of changes showed that this effect persisted in both summer and autumn (Table 3).

Figure 5. dsDNA concentration in the analyzed soil (averages for the entire study period). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

The results obtained for SIR activity (Figure 6A, Table 3) were significantly different between the individual soils and sampling time points. Mean values ranged from 30.37 to 147.788 mg C-CO₂ kg⁻¹ d⁻¹. In fallow soils, this parameter had lower values in soil within the park (42.90 mg) than outside the park (93.90 mg). In contrast, the level of this parameter in field soils from both locations was similar, at 37.45 and 39.63 mg. However, seasonal variation analysis showed that the activity in these soils in summer was also lower within the PNP (Table 3).

Figure 6. Substrate induced respiration (A) and Respiration (B) in the analyzed soil (averages for the entire study period). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

Results for SIR activity in reclaimed soils (Figure 6A) showed that this parameter in soil subjected to 2 years of reclamation reached 30.37 mg, close to the target level observed in soil from the field outside the park (39.63 mg). Ten years of reclamation strongly stimulated this process, reaching the highest level among all soils, i.e., 147.78 mg. Seasonal analysis of SIR activity showed that higher values in both soils were recorded in summer (Table 3).

CO₂ emission reflecting respiratory activity (R) ranged from 1.94 to 4.60 mg C kg⁻¹ d⁻¹ (Table 3). The lowest CO₂ emissions were observed in the cultivated soil within the park and in the R2 soil during the summer sampling, as well as in soil outside the park in autumn. The highest CO₂ emissions (>4 mg) were observed in fallow soils at both locations during the second sampling, and in R10 soil regardless of the sampling time. Based on mean emissions at both sampling time points, the highest CO₂ release occurred in R10 soils and under fallow at both locations, while the lowest was observed in the field soil outside the park and in R2 soil (Figure 6B). The TOC content of R10 soil was two orders of magnitude higher than that of W soil and one order of magnitude higher than CF and R2, which may explain its highest CO₂ emissions despite the low nitrogen content.

Results for dehydrogenase and β-glucosidase activity (Figure 7, Table 3) showed that, similar to microbial abundance and respiration rate, these enzymatic parameters also varied significantly with habitat, location, and sampling time. Mean dehydrogenase activity ranged from 0.26 to 1.17 mg TPF kg⁻¹ h⁻¹ (Figure 7A). In both fallow and field soils, this parameter had lower values within the park (0.27 and 0.40 mg) than outside it (1.17 and 0.59 mg). Mean β glucosidase activity was also lower in the park (11.43 mg PNP kg⁻¹ h⁻¹) than outside it (57.72 mg), but only for fallow soils (Figure 7B). Meanwhile, field soils from both locations showed similar levels of this activity (34.51 and 35.41 mg). However, data from individual sampling time points showed that β-glucosidase activity in autumn was also lower in field soil within the park than outside it (Table 3). Seasonal variation analysis of both enzymatic parameters showed that the differences between locations, i.e., within and outside the PNP, were more significant in autumn (Table 3).

Figure 7. Dehydrogenases (A) and β-glucosidase (B) activity in the analyzed soil (averages for the entire study period). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

Results for soils reclaimed for 2 and 10 years showed that both enzymatic activities were significantly lower than in the target soil, i.e., soil from outside the park, where dehydrogenase activity was 0.59 mg and β-glucosidase activity 35.41 mg (Figure 7A,B). This parameter for mean dehydrogenase activity in both reclaimed soils was at a similar level, 0.26 and 0.32 mg. However, seasonal variation analysis showed that dehydrogenase activity in soil reclaimed for 2 years was lower than in soil subjected to longer reclamation during one season—summer. β-glucosidase activity was clearly dependent on the duration of reclamation (Table 3). In soil reclaimed for 10 years, β-glucosidase activity was significantly higher (31.33 mg) than in soil reclaimed for 2 years (12.51 mg). Seasonal variation analysis of the enzymatic activities in both soils showed that they were generally higher in autumn (Table 3).

Data on phytotoxicity are presented in Figure 8. Analysis of the results showed significant differences between fallow and field soils and their locations only for germination (Figure 8A). The values of this parameter ranged from 78 to 92 germinated seeds. Higher values were observed in both fallow and field soils within the park (92 and 83 germinated seeds, respectively) than in the corresponding soils outside the park (79 and 78 germinated seeds, respectively).

Figure 8. Phytotoxicity parameters: Germination of L. sativum seeds (A) and Root length of L. sativum (B) in the analyzed soil (summer). W—wasteland, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; the vertical lines indicate the standard deviation. Different letters indicate the significance of the differences. 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.

It should be noted that the number of germinated seeds in both reclaimed soils was significantly higher (90 and 87 seeds) than in the target soil, i.e., the field outside the park (78 seeds). Moreover, regardless of the duration of the reclamation period, no significant differences were observed between the two soils.

Another analyzed phytotoxicity parameter related to the initial stage of plant development, i.e., root length after 2 days, showed no significant differences depending on the location of individual sites (Figure 8B). This parameter remained at a similar level both within and outside the park, reaching 1.2 and 1.1 cm, respectively, and for fields, 2.3 and 1.9 cm, respectively. In contrast, the mean root length differed significantly between habitats, with higher values recorded for fields.

In reclaimed soils, root length measured after 2 days was significantly lower (both 1.3 cm) than in soil from the field outside the park (1.9 cm), irrespective of the duration of reclamation (Figure 8B).

4. Discussion

The results for microbial abundance and their biochemical activities showed that soils located within the park, particularly fallow land, differed markedly in this respect from soils outside the park. Bacterial abundance, DNA concentration, enzyme activities, and respiration were lower within the park area (Figure 9). These observations were further supported by principal component analysis (Figure 10A), which grouped the sites, at least at one sampling date, separately, according to their location. This effect was likely associated with the restrictions on soil use applicable within the national park, resulting from the Act of 16 April 2004 on Nature Conservation [54], including, among others, limitations on fertilizer application. The designation of these environmentally valuable areas as a national park resulted in reduced anthropogenic pressure, including agricultural intensification, which in turn allowed the development and maintenance of soil environmental homeostasis [38,55,56].

Figure 9. 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; OB—oligotrophic bacteria, FF—filamentous fungi, CB—cellulolytic bacteria, CF—cellulolytic fungi, DNA—dsDNA concentration, SIR—substrate induced respiration, R—respiration, DEH—dehydrogenases, B-GLU—β-glucosidase.

Figure 10. PCA score plots and corresponding variable loadings and contributions for the first two principal components (PC1 and PC2). (A) PCA including all analyzed parameters, excluding phytotoxicity parameters. (B) PCA based on phytotoxicity parameters, with physicochemical, chemical, and environmental parameters included as supplementary variables. Abbreviations: W—wasteland; CF—cultivated field (in the score plots); s—summer; a—autumn; OB—oligotrophic bacteria; FF—filamentous fungi; CB—cellulolytic bacteria; CF—cellulolytic fungi (in the loading/contribution table); DNA—dsDNA concentration; SIR—substrate induced respiration; R—respiration; DEH—dehydrogenase; B-GLU—β-glucosidase; GERM—germination of L. sativum; RL2—root length of L. sativum after two days.

The lower microbiological and biochemical activity of soils located within the PNP area was likely significantly affected by the lower carbon content, a key nutrient for microorganisms (Table 1). This was likely a consequence of the aforementioned restrictions. Soil bacteria and fungi are heterotrophic microorganisms that show a strong dependence on the carbon content of organic matter [5,57]. In their studies, Wang et al. [58] and Wu et al. [59] confirmed the key role of carbon resources in shaping the structure and activity of the soil microbiome, reporting correlations between TOC content and microbial abundance. Under the conditions of the present experiment, strong correlations were also observed between total bacterial abundance, cellulolytic bacteria, and the TOC content (Figure 11). Correlations with this parameter were also observed for cellulolytic fungi. It should be noted, however, that the abundance of these fungi, as well as filamentous fungi, in contrast to bacteria, was higher in soils within the park than in those outside it. It may be assumed that soil pH was the decisive factor determining their occurrence. Filamentous fungi showed a strong negative correlation with this parameter (Figure 11). As illustrated in Table 1, soils within the park had a lower pH than soils outside it. It is well known that fungi prefer lower pH environments [60,61]. This parameter may have also influenced bacterial abundance, although to a lesser extent than carbon. Total bacterial abundance and the number of cellulolytic bacteria showed positive correlations with pH (Figure 11).

Figure 11. Heatmap displaying the Pearson’s correlation coefficients between physicochemical, chemical, environmental, and microbiological soil properties, as well as between soil phytotoxicity parameters and physicochemical, chemical, and environmental properties. Abbreviations: OB—oligotrophic bacteria, FF—filamentous fungi, CB—cellulolytic bacteria, CF—cellulolytic fungi, DNA—dsDNA concentration, SIR—substrate induced respiration, R—respiration, DEH—dehydrogenases, B-GLU—β-glucosidase, TOC—total organic carbon, TN—total nitrogen, RAIN—rainfall, TEMP—temperature, WHC—water-holding capacity. Significant at * p < 0.05; ** p < 0.01; *** p <0.001, respectively.

Nitrogen content and availability constitute an additional factor that significantly influences microbial activity, including enzymatic processes [62,63]. In the present study, strong positive correlations were observed between dehydrogenase and β glucosidase activities and the content of this nutrient (Figure 11). The ratio of nitrogen to carbon is also critical, as it reflects the substrate balance that regulates microbial processes [64]. Data presented in Table 1 indicate that it was lower in soils within the park, which may have contributed to the inhibition of microbial growth and enzymatic activity in this area. Lower nitrogen content and enzymatic activity may also have been influenced by soil water-holding capacity, as indicated by the strong positive correlations between these parameters (Figure 11). According to the literature, nitrogen content depends on water-holding capacity, affecting the activity and diversity of the soil microbiome [63,65]. Additionally, climatic conditions, namely precipitation and temperature, most likely have a significant impact on its development and activity. This was reflected in the observed negative correlations between bacterial and fungal abundance and meteorological parameters, although they were weaker than for the previously discussed factors (Figure 11). The sensitivity of soil microorganisms to climatic factors has also been confirmed by earlier studies [14,66]. The observed negative correlation between fungal abundance and dehydrogenase activity, alongside the stronger positive correlation between bacterial abundance and this activity, may indicate bacterial dominance in metabolic processes associated with respiration.

It is well known that the plate method is based on the cultivation of microorganisms, which are estimated to represent approximately 1% of the soil microbiome [67]. However, despite their numerous advantages, modern molecular methods have limitations that can affect the reliability of the results obtained—such as the ambiguous differentiation between live and dead cells or issues regarding sample representativeness [68,69,70]. For this reason, soil monitoring employs a comprehensive assessment using this parameter in combination with other indicators of microbial activity, including modern molecular methods. The strong positive correlation observed in the present study between bacterial abundance, determined using the plate method, and DNA concentration indicates that, despite certain limitations, this method provides valuable and reliable information, and its use in soil monitoring is fully justified. Similar conclusions were reached by authors in earlier studies [14]. Moreover, a strong correlation was observed between the isolated DNA and dehydrogenase activity, suggesting that under the conditions analyzed in this work, the genetic material originated primarily from living cells (intracellular DNA). However, studies in this area are not conclusive. Wolińska et al. [71] and Joniec [72] suggested in their studies that the DNA was likely of intracellular origin. Other authors also indicate that DNA isolated from soil may contain a substantial proportion of relic DNA originating from dead cells and, consequently, does not always reflect the active microorganisms [73,74].

Total CO₂ emissions from soil result from the combined activity of plant roots and heterotrophic soil microorganisms [75]; CO₂ emissions measured under laboratory conditions from soil samples, including those with added glucose, reflect only the activity of heterotrophic bacteria and fungi. As reported by De Dato et al. [75], this heterotrophic respiration varies depending on the ecosystem and abiotic and biotic factors. Previous studies have shown that land use, together with climate, land cover, vegetation, and nutrient status, significantly influences soil greenhouse gas fluxes [3,25]. Cultivated soils generally produce higher CO₂ emissions than wastelands due to agricultural practices such as fertilization and tillage [25,76]. An experiment comparing tillage systems demonstrated higher CO₂ emissions directly after autumn ploughing than after deep or shallow cultivation and no-tillage treatments [76]. Soil disturbance accelerates organic matter decomposition through disruption of soil structure, which increases CO₂ release.

In the present study, however, wastelands emitted more CO₂ than cultivated fields, and the sampling period exerted a stronger effect on emissions than land use. In most cases, higher CO₂ fluxes were recorded in autumn despite lower temperature and precipitation than in summer (Figure 2). The soils differed in carbon and nitrogen contents. Wastelands contained approximately one order of magnitude less carbon and more nitrogen than cultivated and reclaimed soils (Table 2). Consequently, the soils varied in the C/N ratio, which influences soil respiration. The differences in CO₂ emissions and SIR observed between sites located within and outside the park likely resulted from the sensitivity of the microorganisms responsible for CO₂ production to various environmental factors. Correlations were observed between the abundance of these microorganisms and temperature, moisture, TOC and TN content, and soil pH, while respiration was directly correlated with pH and TOC (Figure 11). Similar relationships have also been observed by other authors [58,77]. The differences recorded in this study between basal respiration (R) and glucose-induced soil respiration (SIR) were likely caused by the supply of an easily available carbon source to soil microorganisms, which enhanced their activity. At the same time, glucose addition was reported to reduce the nitrogen needed for maximum CO₂ release, suggesting that the availability of labile carbon decreased microbial nitrogen demand, which was influenced by the soil’s initial nitrogen content [78]. This may partly explain the differing responses of natural and glucose-induced CO₂ emissions observed in our study. It should be noted, however, that despite these differences, soil respiration (R) showed a positive correlation with SIR, suggesting that in the present experiments, CO₂ was derived primarily from microbial, rather than abiotic, processes. Measuring soil CO₂ emissions provides valuable information on the rate of carbon mineralization in the soil. Consequently, it represents an important aspect of soil monitoring, enabling interventions to limit the rate of carbon loss from soils when required [79].

Soil phytotoxicity in fields and fallow land also varied depending on location, with higher parameter values recorded in soils in the park. Principal component analysis (Figure 10B), consistent with the results for microbial activity, indicated a clear separation of fallow soils from the remaining sites. These observations indicate that soils within the PNP likely offer more favorable conditions for the early stages of plant development, including germination and seedling root growth. Negative correlations were observed between root growth and both nitrogen content and soil pH (Figure 11). These observations suggest that the lower nitrogen content and reduced pH in protected soils, particularly in fallow land (Table 1), may have been more favorable for seedling root growth. For the germination process, however, water-holding capacity appears to be a key factor, as this parameter was negatively correlated with germination (Figure 11). The results in this aspect are not conclusive, as other authors reported a positive correlation between phytotoxicity parameters related to the initial growth of L. sativum and nitrogen content, pH, or moisture [80,81].

Both 2- and 10-year reclamation with gangue did not cause an increase in soil microbial activity close to the target activity level, i.e., that observed in soil from the field outside the park (Figure 9). These observations are confirmed by the principal component analysis (PCA) presented in Figure 12A, which clearly indicates separate clustering of individual soils, i.e., soil reclaimed for 2 years and for 10 years, as well as soil from outside the park. This distribution confirmed that the activities analyzed at individual sites (at both time points) differed significantly. This variation was determined by the first principal component (PC1) in 49.19% and in 27.06% by the second principal component (PC2). It should be noted that long-term reclamation with gangue (10 years) stimulated higher microbiological activity and, consequently, reached values closer to those of the target soil, i.e., from the field outside the park. These observations indicate that reclamation with gangue requires a longer period of time. At the same time, it should be emphasized that the number of scientific reports directly referring to the long-term impact of waste rock on the biological properties of soils is limited, which further highlights the importance and relevance of the current research [29,38,82]. However, it is noteworthy that the highest CO₂ volume was emitted from soil that had been reclaimed for 10 years, both from non-induced and glucose-induced soil. This is a negative phenomenon from the greenhouse effect perspective, as CO₂, alongside N₂O and CH₄, plays a significant role in intensifying this adverse process [28]. In addition, increased CO₂ emissions may result in carbon losses from the soil [79]. The effect observed in the present experiments may have been related to the highest content of this element (TOC) at this site (Table 1). This soil was characterized by the highest CO₂ emissions, highest C content, and low nitrogen content. The ratio of these two elements (C/N) could therefore play a key role in emission intensification of the analyzed gas. This is confirmed by earlier studies indicating the significant importance of the C/N ratio in regulating soil microbial activity [83,84]. The high microbial activity observed at the 10-year reclamation site may be associated with its relatively high C/N ratio, reflecting a high supply of organic C relative to N. Under these conditions, C-rich substrates may provide sufficient energy to sustain intensive microbial activity, despite the relatively low N concentration. Positive relationships between soil C/N ratio and microbial basal respiration and microbial biomass have previously been reported [85]. Low N availability may promote microbial N mining, whereby microorganisms decompose N-rich organic matter to meet their N demand, potentially sustaining high respiration rates [86]. However, microbial activity is also influenced by substrate quality, C availability, and the degree of nutrient limitation. Beneficial changes in biological activity, accompanied by increased CO₂ emissions, suggest that reclamation using gangue alone requires additional measures to retain carbon, an important nutrient, in the soil. One approach involves increasing soil nitrogen content, e.g., by applying nitrogen fertilization in combination with gangue or by introducing leguminous plants into the cultivation system [87,88]. Establishing a favorable C:N ratio in the soil promotes carbon stratification and prevents its loss [64,89]. In forest ecosystems, nitrogen addition positively affected soil organic carbon accumulation by increasing carbon content in microbial biomass, suggesting that reduced nitrogen limitations stimulated microbial growth and subsequent incorporation of carbon into the soil [64].

Figure 12. Principal component analysis (PCA), score plot (A) and loading plot (B) of the analyzed parameters (excluding phytotoxic parameters) with heavy metal content in soil from the PNP field and in soil from reclaimed areas. The corresponding loadings and contributions of the analyzed variables to the first two principal components (PC1 and PC2) are presented below the plots. Abbreviations: OB—oligotrophic bacteria, FF—filamentous fungi, CB—cellulolytic bacteria, CF—cellulolytic fungi, DNA—dsDNA concentration, SIR—substrate induced respiration, R—respiration, DEH—dehydrogenases, B-GLU—β-glucosidase, TOC—total organic carbon, TN—total nitrogen, RAIN—rainfall, TEMP—temperature, WHC—water-holding capacity, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; s—summer, a—autumn.

According to the literature, gangue is a waste material that can introduce a certain load of heavy metals into the soil environment [90]. However, in the present study, the concentrations of selected heavy metals in the reclaimed soils remained within the permissible limits established by Polish and European regulations [91,92]. However, their levels in these soils were higher than in the field soil (Table 2). Analysis of the results presented in Figure 12B shows that As and Hg were negatively correlated with dehydrogenase activity, which may indicate a potential toxic effect of these heavy metals on this enzymatic activity. The more favorable conditions in soil reclaimed for 10 years, compared with soil reclaimed for 2 years, i.e., higher pH and organic carbon content, may have mitigated the potential adverse effects of heavy metals on the growth and activity of soil bacteria and fungi. It is well known that the bioavailability of heavy metals is influenced, among others, by soil pH. Additionally, organic matter plays a protective role against their toxic effects [93,94]. Phytotoxicity results showed significantly lower seedling root growth in soils reclaimed with gangue, both in the initial and later stages of reclamation. Expanding soil monitoring to include ecotoxicological tests allows for a more precise assessment of potential risk. Moreover, ecotoxicological analysis considers possible interactions between contaminants in both direct and indirect contexts of soil physicochemical factors [95]. Principal component analysis presented in Figure 13A revealed significant differences between reclaimed sites and the field outside the park. This distribution confirms that the analyzed activities differed significantly between the individual sites. A negative correlation was observed between Hg and As concentrations and seedling root growth (Figure 13B). These findings, as for dehydrogenase activity, indicate that heavy metals may have exerted a toxic effect on this parameter.

Figure 13. Principal component analysis (PCA), score plot (A) and loading plot (B) of the analyzed phytotoxic parameters with heavy metal content in the soil from the PNP field and in the soil from the reclaimed areas. The corresponding loadings and contributions of the analyzed variables to the first two principal components (PC1 and PC2) are presented below the plots. Abbreviations: GERM—germination of L. sativum, RL2—root length of L. sativum after two days, TOC—total organic carbon, TN—total nitrogen, RAIN—rainfall, TEMP—temperature, WHC—water-holding capacity, CF—cultivated field, R2—reclaimed land 2 years, R10—reclaimed land 10 years; s—summer, a—autumn.

In summary, although the concentrations of heavy metals in soils treated with gangue did not exceed permissible levels (Table 2), these elements may have exerted toxic effects on soil organisms during the initial years and, with respect to phytotoxicity, potentially over a longer period.

These observations indicate a strong need to monitor soils with gangue introduction, regardless of its chemical composition. Assessment of specific microbiological processes related to carbon transformations in soil will enable the implementation of improved soil management practices focused on improving carbon use efficiency and reducing greenhouse gas emissions.

5. Conclusions

The results showed that the analyzed activities differed significantly by location, namely between sites within the national park and those outside its boundaries. The differences were also more pronounced in fallow land soils than in cultivated field soils. Bacterial growth, dsDNA concentration, as well as respiratory and enzymatic activities in both fallow and cultivated field soils were generally lower in the park, with the strongest effect observed in fallow soils. In contrast to bacteria, the growth of both filamentous and cellulolytic fungi was greater in the fallow and cultivated field soils located within the park than in those outside it. Phytotoxicity analysis indicated that soils within the national park provided favorable conditions for early plant development, including germination and root growth. Thus, the results allowed to verify and confirm the hypothesis regarding the protective role of the park in maintaining soil environmental homeostasis and improving conditions critical for early plant development.

The results concerning the use of gangue for reclamation showed that this process was not effective. In most cases, the levels of the analyzed parameters in soils reclaimed using this waste remained below those recorded in the target soil, i.e., the field outside the park. However, this effect depended on the duration of reclamation. Almost all activities were lower in soil reclaimed for 2 years than in the target soil. Longer reclamation (10 years) proved more effective, but only for some parameters (abundance and respiration). However, this soil also released the highest amount of CO₂, which is undesirable from the perspective of global climate change. The phytotoxicity results for soils reclaimed with the waste were inconclusive and indicated a potential negative effect of gangue on the early stages of plant development. Accordingly, the hypothesis regarding the suitability of gangue for reclamation was only partially confirmed. Thus, the present results indicate the need for further research into safer and more effective methods of reclamation using gangue, taking into account the simultaneous increase in soil C accumulation and reduction in phytotoxicity.

The current study provided valuable information on the role of microorganisms in the biogeochemical carbon cycle in environmentally valuable areas that are simultaneously exposed to various forms of anthropogenic pressure. The results demonstrated the effectiveness of the implemented conservation measures. Moreover, they indicate the need to reassess soil reclamation based solely on gangue, to incorporate additional remedial treatments, and to conduct further research in this field.