Changes in Stabile Organic Carbon in Differently Managed Fluvisol Treated by Two Types of Anaerobic Digestate

Abstract

Biogas and anaerobic digestion has begun to be considered an important renewable and sustainable energy source. The sustainable development of the anaerobic digestion process depends largely on the ability to manage large amounts of by-products generated during the biogas production process. We hypothesized that the use of digestate increases the accumulation of C in stable forms. We supposed that the effect of digestate on soil depends on the land-use system, leading to different stratifications of C. The main task of our research was to ascertain changes in the amount of stabile organic carbon (SOCstabile) in digestate-treated soils. Two field experiments were performed using the same design in 2019–2020. We studied the fertilization effects of digestate on Fluvisol. Fertilization: control; separated liquid digestate 85 kg ha⁻¹ N and 170 kg ha⁻¹ 170 N; separated solid digestate 85 kg ha⁻¹ N and 170 kg ha⁻¹ N. A randomized experimental design with three field replicates was used. In terms of carbon stabilization in Fluvisol, soil used for grassland showed an advantage over the arable soil. The study showed that digestate, especially solid digestate, contributes to C accumulation and stabilization in the soil.

1. Introduction

The production in all agricultural sectors and in the food industry generates large amounts of biodegradable waste, which must be managed responsibly. This waste has a high potential for biogas production, and its use in this way brings additional economic, environmental, and climate benefits [1,2,3,4]. Anaerobic digestion is a biological process during which, under anaerobic conditions, microorganisms degrade organic matter into simpler components, and in this way, biogas is produced [5,6]. Biogas is a mixture of methane, carbon dioxide, and a small amount of hydrogen sulfide, ammonia, and moisture [7]. Biogas is a promising and renewable energy source for electricity and heat production, and, if properly purified to biomethane, it can be injected into the natural gas network or used in vehicles as fuel [2,8,9]. Increasing biogas use is one of the European Commission’s commitments to meet its climate objectives and reduce energy dependence on foreign countries [10].

However, the sustainable development of the anaerobic digestion process depends largely on the ability to manage large amounts of by-products generated during the biogas production process [11,12]. With the increase in biogas production seen in recent years, the amount of digestate has increased [13]. Digestate can improve soil quality, stimulate crop yields, and even positively influence soil bacteria growth [14,15]. Depending on the technological application possibilities, anaerobic digestate (AD) could be used whole or separated into solid and liquid phases [4,16,17,18].

All types of digestate are reservoirs of organic carbon and plant nutrients, but they have quantitative and qualitative differences: solid digestate contains 38–75% of highly stable organic matter and a low NH4-N/total-N ratio; liquid digestate has a low organic matter concentration and a high NH4-N/total-N ratio. The use of solid digestate as a soil amendment can aid soil carbon sequestration and improve soil physical properties. Liquid digestate is more suitable for use as a biofertilizer [17,19,20]. Therefore, studies in various fields on the chemical composition of solid and liquid digestate fractions are important, with these having shown that liquid and dry digestate fractions have different effects on soil [18,19,21].

Soil organic carbon (SOC) plays a critical role in determining soil properties. Maintaining SOC stock at equilibrium and increasing SOC content is important for the following global processes: the provision of food and energy for soil ecosystems, improving nutrient availability for plants, supporting the productivity of aboveground plants and animals, ensuring chemical reaction, transformation, and exchange for optimal nutrient and water availability, improving soil physical structure for optimal provision and exchange of air, water, and gases, and mitigating climate change effects by carbon (C) sequestration and offsetting greenhouse gases emissions [22,23]. However, as is known, SOC is affected by various natural and anthropogenic perturbations, such as wildfire, climate change, land-use change, and pollution [24]. Increasing carbon (C) storage in soil is a key aspect of climate change mitigation strategies and requires an understanding of the impacts of land management on soil C cycling [25]. Increasing plant inputs to soils, whether from living plants or their organic residues, promote the growth of C flow [26]. Digestate is also a source of organic carbon and plant nutrients and can contribute to the accumulation of soil organic matter [19,27,28,29]. Most studies reporting on soil C sequestration focus on measurements of total SOC, but the quality of SOM can also significantly influence the lifetime of carbon storage in the soil [30,31]. An increase in SOC content because of a reduction in tillage intensity is accompanied by an increase in the amount of humified carbon content [32]. Carbon sequestration must be considered a process of C stabilization and not just a simple accumulation of organic matter in the soil [25]. One of the more important new effects on soil properties is the expected changes in SOC using digestate, contributing to the long-term accumulation of stable forms of carbon and in turn leading to C sequestration.

According to Yang et al., in cropped soils, SOC is usually less vertically stratified than in soils under natural ecosystems [33]. According to Blonska et al., their research confirmed the decreasing trend of soil organic carbon content with increasing soil depth, in both woodland and agricultural soils [25]. Based on the results of previous extensive studies, the stratification ratio (SR) is an efficient indicator of the dynamics of SOC sequestration and soil quality [34,35,36], and an SR2 (0–5:20–30 cm) >2 has been reported to indicate a distinct improvement in SOC sequestration and soil quality in ecological restoration on a hilly relief [36].

We hypothesized that the use of digestate increases the accumulation of C in stable forms and contributes to the reduction of C emissions. We also hypothesized that the effect of the digestate on the soil depends on the land-use system, leading to different stratifications of C.

The main task of this research was to ascertain changes in the amount of stabile organic carbon in anaerobic-digestate-treated soils.

2. Materials and Methods

2.1. Study Area

The field experiment was performed near Surviliskis, Kedainiai District on the first alluvial terrace of the Nevezis river, which is in the middle part of Central Lithuania’s lowland, in Fluvisol (Figure 1).

The study area is located within the agro-climatic region, where the sum of active temperatures (t > 10 °C) is 2190–2250 °C in the air and 2500–2600 °C in the soil. In April 2019, the average air temperature was +8.9 °C, which is 2.9 °C above the perennial average. April was very dry; there was no precipitation at all. In May, the average temperature was +12.9 °C, which is 0.5 °C higher than the perennial average. The amount of precipitation in May was similar to the perennial average (51.4 mm). However, the lack of moisture in the soil was obvious. In the spring of 2020, meteorological conditions were like those prevailing in 2019—air temperatures in April were higher than the average perennial. However, May was much cooler than usual, recording an average monthly temperature of only 10.6 °C. Rainfall during this month was close to the perennial average. Two land-use systems were investigated: semi-natural grassland, 10 years old, prior to the experiment used for forage; the crop rotation field (cereals were grown). In the crop rotation field, in 2019, a mixture of oat (Avena sativa L.) and vetch (Vicia sativa L.) was grown, and in 2020, winter triticale (Triticum × secale) was grown. The conventional soil tillage method in the crop rotation field was used. Soil pH values (6.2–6.5) were favorable for agricultural crop growth. Prior to the experiment in April 2018, the topsoil (0–30 cm) characteristics were as follows: humus content between 2.48 and 2.02%; plant available phosphorus (P₂O₅) and potassium (K₂O) were 464 and 508 mg kg⁻¹, respectively; the subsoil (30–50 cm) pH was about 6.8 and humus content was 0.43%.

The semi-natural grassland in the experimental site was also studied. The 170 kg ha⁻ N fertilization rate was selected as the maximum possible fertilization rate based on the EU Nitrates Directive [37], and the 85 kg ha⁻¹ N fertilization rate was selected as the lower fertilization rate. Five fertilization treatments were investigated: (1) control (no fertilizer was applied); (2) separated liquid digestate 85 kg ha⁻¹ N (85N LD); (3) separated liquid digestate 170 kg ha⁻¹ N (170N LD); (4) separated solid digestate 85 kg ha⁻¹ N (85N SD); (5) separated solid digestate 170 kg ha⁻¹ N (170N SD). The application rates of LD and SD were calculated based on the total Kjeldahl nitrogen (TKN) content in the digestate. A randomized experimental design with three field replicates was used, and the plots were 2 × 3 = 6 m². Dry and liquid digestate was spread on the field manually. Fertilizers were applied in April of 2019 and 2020.

2.2. Digestate Sampling and Analyses

Digestate sampling from a biogas power plant, located in Lithuania, Krekenava, was carried out in the spring of the experimental years 2019 and 2020. For fertilization, the samples of anaerobic digestion by-product digestate were removed from the reactor and both output streams (solid and liquid fractions). Approximately 5 kg of separated SD and 5 L of separated LD were collected and delivered to the laboratory. Digestate samples were stored at −20 °C until chemical analysis. The digestate chemical composition was previously documented by Slepetiene et al. [38].

2.3. Soil Sampling and Analyses

In Fluvisol, located in Kedainiai District Municipality, Lithuania, the influence of two types of digestate on the soil in two land-use systems, semi-natural grassland and a crop rotation field where cereals were grown, was researched. Soils were sampled from 0–10, 10–20, 20–30, and 30–40 cm 5–6 weeks after fertilization. Five subsamples per plot were taken randomly using a steel auger. Soil chemical analyses were carried out at the Chemical Research Laboratory of the Institute of Agriculture (LAMMC). Prior to chemical analyses, the samples were crushed and sieved through a 2-mm sieve. For the analysis of SOC and carbon fractions, an aliquot of the samples was passed through a 0.25-mm sieve. The content of SOC was determined according to the Tyurin method modified by Nikitin [39], with spectrophotometric measurement carried out at a wavelength of 590 nm using glucose as a standard after wet combustion. Soil pH was determined by the potentiometric method in 1 M KCl (1:2.5, w:v). The carbon of mobile humic substances (MHS) was extracted with 0.1 M NaOH. The MHS were determined after wet combustion by spectrophotometric measurement analogous to that for SOC [38,40]. The amount of stabile soil organic carbon was identified as the difference between SOC and MHS.

2.4. Statistical Analysis

Data were evaluated according to Duncan’s multiple range tests at the probability level of p < 0.05 and correlation-regression analysis by the software package SAS version 7.1 (SAS Institute Inc., Cary, NC, USA). Data were subjected to a one-way analysis of variance (ANOVA) according to the treatment structure. Vertical error bars display the value of standard errors.

3. Results and Discussion

3.1. The Effect of Digestate-Treated Soils on the Stratification Ratio of Soil Organic Carbon

The stratification ratios of soil organic carbon (SOC) in digestate-treated soils under different land-use systems in 2019–2020 are shown in

Table 1. Stratification ratios of SOC in digestate-treated soils under two land-use systems in 2019 and 2020.

Land Use, Year	Depth, cm	Fertilization
		No Fertilizer	85 N Solid	85 N Liquid	170 N Solid	170 N Liquid
SOC (g kg−1)
Crop rotation field, 2019	0–10	11.35 ± 0.11	13.66 ± 1.28	11.39 ± 0.14	14.22 ± 0.25	11.99 ± 0.10
	10–20	10.54 ± 0.20	10.93 ± 0.75	10.75 ± 0.37	11.74 ± 0.32	12.60 ± 0.26
	20–30	3.66 ± 0.90	4.38 ± 0.88	6.27 ± 1.00	5.64 ± 0.81	5.52 ± 1.69
	30–40	2.28 ± 0.53	2.31 ± 0.49	3.68 ± 0.77	2.80 ± 1.07	3.28 ± 0.56
Pr > F		<0.0001	<0.0001	<0.0001	<0.0001	0.0002
Grassland, 2019	0–10	18.26 ± 3.08	15.13 ± 0.88	15.71 ± 1.61	15.54 ± 1.61	16.11 ± 1.51
	10–20	9.30 ± 0.58	11.00 ± 1.03	11.87 ± 0.82	12.03 ± 0.66	10.78 ± 1.48
	20–30	3.98 ± 0.68	4.50 ± 0.89	5.40 ± 0.64	4.29 ± 0.67	4.32 ± 0.99
	30–40	1.79 ± 0.14	1.56 ± 0.51	2.23 ± 0.14	1.79 ± 0.21	2.36 ± 0.41
Pr > F		<0.0001	<0.0001	<0.0001	<0.0001	0.0001
Crop rotation field, 2020	0–10	13.18 ± 0.08	13.38 ± 0.19	12.56 ± 0.16	13.34 ± 0.52	12.67 ± 0.59
	10–20	12.23 ± 0.15	12.70 ± 0.49	11.78 ± 0.22	12.78 ± 0.20	12.27 ± 0.61
	20–30	7.52 ± 0.81	7.63 ± 0.94	5.82 ± 2.41	8.14 ± 0.76	7.16 ± 2.50
	30–40	2.94 ± 0.98	3.97 ± 1.42	2.69 ± 0.06	3.92 ± 0.27	3.44 ± 1.34
Pr > F		<0.0001	0.0002	0.0011	<0.0001	0.0063
Grassland, 2020	0–10	14.82 ± 1.27	13.41 ± 1.26	14.90 ± 0.87	15.09 ± 1.26	15.71 ± 1.01
	10–20	10.57 ± 0.94	8.29 ± 0.46	9.39 ± 1.27	10.45 ± 1.45	10.03 ± 0.87
	20–30	4.74 ± 0.69	3.15 ± 0.39	4.62 ± 1.55	3.07 ± 0.34	4.40 ± 0.84
	30–40	1.90 ± 0.19	1.83 ± 0.31	1.90 ± 0.42	1.94 ± 0.56	2.28 ± 0.27
Pr > F		<0.0001	<0.0001	0.0002	<0.0001	<0.0001
Stratification ratio, SOC
Crop rotation field, 2019	(0–10:10–20)	1.08 ± 0.03	1.25 ± 0.05	1.06 ± 0.02	1.21 ± 0.02	0.95 ± 0.03
	(0–10:20–30)	3.45 ± 0.71	3.26 ± 0.40	1.92 ± 0.34	2.63 ± 0.36	2.93 ± 1.24
	(0–10:30–40)	5.60 ± 1.37	6.20 ± 0.66	3.45 ± 0.85	6.52 ± 1.88	3.88 ± 0.67
Pr > F		0.0323	0.0007	0.0479	0.0351	0.1051
Grassland, 2019	(0–10:10–20)	1.65 ± 0.06	1.40 ± 0.14	1.32 ± 0.05	1.29 ± 0.07	1.51 ± 0.07
	(0–10:20–30)	4.08 ± 0.74	3.65 ± 0.74	2.92 ± 0.14	3.76 ± 0.53	4.05 ± 0.72
	(0–10:30–40)	8.64 ± 0.75	11.72 ± 3.11	7.00 ± 0.29	9.03 ± 1.81	7.05 ± 0.68
Pr > F		0.0005	0.0171	0.0001	0.0064	0.0014
Crop rotation field, 2020	(0–10:10–20)	1.08 ± 0.02	1.06 ± 0.03	1.07 ± 0.02	1.04 ± 0.03	1.03 ± 0.03
	(0–10:20–30)	1.79 ± 0.19	1.81 ± 0.22	2.90 ± 0.91	1.66 ± 0.11	2.48 ± 1.06
	(0–10:30–40)	5.51 ± 1.62	4.40 ± 1.55	4.68 ± 0.16	3.44 ± 0.29	4.60 ± 1.20
Pr > F		0.0328	0.0875	0.0088	0.0002	0.0873
Grassland, 2020	(0–10:10–20)	1.40 ± 0.05	1.64 ± 0.23	1.65 ± 0.26	1.54 ± 0.37	1.61 ± 0.25
	(0–10:20–30)	3.21 ± 0.32	4.43 ± 0.76	3.80 ± 0.86	5.04 ± 0.73	4.04 ± 1.23
	(0–10:30–40)	7.93 ± 1.01	7.91 ± 1.78	8.61 ± 1.94	9.30 ± 2.74	7.23 ± 1.44
Pr > F		0.0007	0.0213	0.0185	0.0438	0.0313

Note: ±standard error of concentration and stratification ratio of soil organic carbon.

.

Our results show that the fertilization approach increased the SOC concentration in the soil regardless of the different land uses and years. Separated liquid digestate from the Krekenava biogas plant had a low total solids (TS) content (2.3–3.6%); separated solid digestate had a higher TS content (29.7–31.3%). The separated solid digestate had 0.96–0.97%, and the separated liquid digestate had 42.6–52.7% of organic C [38]. The SOC concentrations decreased with soil depth and had a differential distribution among digestate-treated soils in different land uses. The SOC content within the land use amounted to between 2.31 and 14.22 g kg⁻¹ in the crop rotation field and to between 1.56 and 18.26 g kg⁻¹ in the semi-natural grassland (Table 1). The stratification ratios of SOC increased with soil depth. The SOC in the surface to lower depth ratio (0–10:30–40) was >6 in the crop rotation field and >11 in the grassland (Table 1). The same trend has been found in studies by other researchers [34,41].

3.2. Effect of Two Fractions of Digestate on the Stabile Soil Organic Carbon (SOCstabile) Pool

Carbon stabilization in soil is the result of interaction between the chemical and physical mechanisms of protection, and the dominance of the mechanisms depends not only on the long-term constant characteristics of soil but also on the properties, which can be partly influenced by human activities [42]. In agriculture, soil management practices can change soil conditions that influence the process of soil organic carbon preservation. The intensification of SOC loss or accumulation and soil quality depends on the C input [41,43].

Figure 2 shows different levels of SOCstabile in 0–10, 10–20, 20–30, and 30–40 cm soil layers in the crop rotation field both treated and not treated by anaerobic digestate. The greatest significant changes in the content of SOCstabile occurred in the soil layer of 0–10 cm. Fertilizing with the maximum SD rate, SOCstabile increased from 8.61 g kg⁻¹ in the control treatment, up to 11.16 g kg-1 when fertilizing at the maximum rate, and up to 10.58 g kg⁻¹ at a lower rate.

With the use of solid digestate (SD), significant increases in SOCstabile when using both fertilization rates (85 and 170 kg ha⁻¹) were observed. In grassland soil, similar trends in changes in stabilized organic carbon content were identified. In this soil, the initial level of SOCstabile was already higher, so the changes in the soil treated by digestate were weaker (Figure 3).

In the crop rotation field in SD treatments during both years of research, higher levels of TS and organic C were inserted, which led to an increase in the content of Cstabil in the soil layer of 0–10 cm (Figure 2 and Figure 4). After fertilizing with SD with a higher and lower fertilization rate, in the second year of the experiment in the crop rotation field, an increase in SOCstabile in the layer of 10–20 cm was established, as well as in the soil layer of 0–40 cm. This was influenced by tillage after harvesting, leading to the mixing of both top layers of soil (Figure 4). The changes in SOCstabile in the crop rotation field also were influenced by plants grown in both years and by the agricultural technologies used for them.

Unlike the crop rotation field, grassland soil layers were not mixed, and the higher accumulation of SOCstabile in the upper layer of soil (0–10 cm) remained (Figure 4). Thus, in grassland soil, all treatments formed higher levels of SOC compared to the crop rotation field. Treating by digestate maintained these higher SOCstabile levels compared to the crop rotation field, and even increased the levels even higher. Figure 3 and Figure 5 show that in the grassland soil in both years of study, the maximum accumulation of SOCstabile in a layer of 0–10 cm (10.11–10.13 g kg⁻¹) was obtained by fertilizing with a maximum rate of 170 kg ha⁻¹ SD, as well as LD, which was analogous, being only slightly less.

Both the liquid and solid fractions of digestate contain valuable components for plant growth during the vegetation period [38]. However, the LD fraction contains less TS compared to the SD fraction. As a result, the LD fraction has more potential to migrate into deeper soil layers compared to the SD fraction. This is in line with the data obtained by other researchers. For example, it was concluded that the solid fraction of digestate can be used as an organic amendment due to its high OC content and its high stability once it is processed through anaerobic digestion [44].

3.3. The Effect of Digestate-Treated Soils on the Stratification Ratio of Stabile Soil Organic Carbon Pool

Stratification ratios of SOCstabile in digestate-treated soils under different land-use systems in 2019–2020 are shown in

Table 2. Stratification ratio of stabile soil organic carbon (SOCstabile) ± standard error in digestate-treated soils under two land-use systems in 2019 and 2020.

Land Use, Year	Depth, cm	Fertilization
		No Fertilizer	85 N Solid	85 N Liquid	170 N Solid	170 N Liquid
Stratification Ratio, SOCstabile
Crop rotation field, 2019	(0–10:10–20)	1.10 ± 0.02	1.31 ± 0.06	1.11 ± 0.04	1.29 ± 0.04	0.92 ± 0.03
	(0–10:20–30)	3.96 ± 0.43	3.10 ± 0.32	1.98 ± 0.35	2.64 ± 0.39	2.60 ± 1.04
	(0–10:30–40)	5.18 ± 1.41	5.45 ± 0.62	3.05 ± 0.72	5.92 ± 1.66	3.50 ± 0.59
Pr > F		0.0362	0.011	0.0656	0.0394	0.0941
Grassland, 2019	(0–10:10–20)	1.78 ± 0.04	1.42 ± 0.24	1.49 ± 0.11	1.35 ± 0.10	1.56 ± 0.11
	(0–10:20–30)	3.91 ± 0.79	3.48 ± 0.84	2.84 ± 0.08	3.60 ± 0.46	3.87 ± 0.90
	(0–10:30–40)	7.36 ± 1.01	13.38 ± 4.55	5.90 ± 0.54	7.97 ± 2.20	5.42 ± 0.41
Pr > F		0.0051	0.0406	0.0002	0.0295	0.0088
Crop rotation field, 2020	(0–10:10–20)	1.09 ± 0.04	1.07 ± 0.04	1.07 ± 0.02	1.07 ± 0.01	1.01 ± 0.02
	(0–10:20–30)	1.74 ± 0.14	1.80 ± 0.23	2.77 ± 0.83	1.65 ± 0.11	2.30 ± 0.92
	(0–10:30–40)	5.26 ± 1.66	4.15 ± 1.44	4.21 ± 0.08	3.42 ± 0.34	4.14 ± 1.04
Pr > F		0.0451	0.0916	0.0106	0.0005	0.0841
Grassland, 2020	(0–10:10–20)	1.35 ± 0.03	1.56 ± 0.21	1.57 ± 0.27	1.60 ± 0.31	1.53 ± 0.23
	(0–10:20–30)	2.90 ± 0.39	3.89 ± 0.79	3.32 ± 0.75	5.19 ± 0.83	3.78 ± 1.20
	(0–10:30–40)	6.30 ± 0.86	6.07 ± 1.58	6.46 ± 1.27	7.57 ± 1.88	5.43 ± 1.05
Pr > F		0.0018	0.0561	0.0193	0.0342	0.0649

. SOCstabile in the surface to lower depth ratio (0–10:30–40 cm) in the crop rotation field in 2019 was 5.45 ± 0.62 when fertilizing by the 85N rate and 5.92 ± 1.66 when fertilizing by the 170N rate of the solid digestate. In grassland soil, the stratification ratios were, respectively, 13.38 ± 4.55 and 7.97 ± 2.20 (Table 2). In 2020, higher stratification ratios (0–10:30–40 cm) in grassland soil compared to the crop rotation field were also observed. According to Franzluebbers, high stratification ratios of soil C and N pools are good indicators of soil quality, independent of soil type and climatic regime, because ratios >2 would be uncommon under degraded conditions [45]. In our study, the stratification ratios of SOCstabile increased with depth. This indicates that Cstabile stratification ratio values of >2 in deeper soil layers show favorable conditions for protecting the soil from degradation by increasing C stability.

We conclude that great attention must be paid not only to the assessment of the total amount of SOC when fertilizing with a new bio-fertilizer, digestate, but also to SOC’s individual fractions, especially the stabile SOC.

Significant positive linear regressions among SOCstabile for 0–10:10–20 cm and SOC for 0–10:10–20 cm are shown in

Table 3. Regression equations among stratification ratios for 0–10:10–20 cm of stabile soil organic carbon (SOC_stabile) and soil organic carbon (SOC) in different land-use systems in 2019 and 2020.

Land Use, Year	Axis		Equations	R2	Significant Level
	X	Y
Crop rotation field, 2019	SOCstabile	SOC	Y = 0.74X + 0.26	0.97	<0.0001
Grassland, 2019	SOCstabile	SOC	Y = 0.66X + 0.43	0.79	<0.0001
Crop rotation field, 2020	SOCstabile	SOC	Y = 0.61X + 0.41	0.59	0.0008
Grassland, 2020	SOCstabile	SOC	Y = 1.02X + 0.01	0.89	<0.0001

. The relationships between stratification ratios for 0–10:10–20 cm of SOCstabile and SOC in different land-use systems in 2019–2020 were significantly (p < 0.001) positive (Table 3). R² varied in the range of 0.59–0.97 in the crop rotation field and the range of 0.79–0.89 in grassland soil, and this illustrates the strong relations between these indicators for the 0–10:10–20 cm soil layer.

The final results of many scientific studies are the proposals of a new technical device, principle, or a new method [46]. As the result of our experiments, a new way to assess the sustainability of carbon in the soil by determining its stabile carbon fraction was proposed.

4. Conclusions

The effect of digestate on soil depends on the digestate type and the land-use system, which also leads to different stratifications of C. In terms of carbon stabilization in Fluvisols, soil used for grassland has shown an advantage over the arable soil used for crop rotation. This study showed that digestate, especially solid digestate, contributes to C accumulation and stabilization in the soil. The Cstabile stratification ratio values of >2 in deeper soil layers show favorable conditions for protecting the soil from degradation by increasing C stability. Further research is needed to investigate the influence of long-term fertilization by digestate on soil C changes and C stability in different soil types and various climatic conditions.