Effects of Combined Application of Solid Pyrolysis Products and Digestate on Selected Soil Properties of Arenosol and Plant Growth and Composition in Laboratory Experiments

Abstract

Biochars as soil amendments have been reported to improve soil properties and may have an important role in the mitigation of the consequences of climate change. As a novel approach, this study examines whether biochar and digestate co-application can be utilized as cost-effective, renewable plant nutrients. The effects of two types of biochar—wood chip biochar (WBC) and animal bone biochar (ABC), applied alone or in combination with an anaerobic digestate—on soil physicochemical properties, on the levels of selected elements, and on growth yields of ryegrass were studied in laboratory experiments. Most parameters were significantly affected by the treatments, and the investigated factors (biochar type, application rate, and the presence of digestate), as well as their interactions, were found to have significant effects on the characteristics investigated. The easily soluble phosphorus content (AL-P₂O₅) of the soil increased in all WBC and ABC biochar treatments, and the presence of digestate caused a further increase in AL-P₂O₅ in the case of anaerobic digestate-supplemented ABC treatment (ABCxAD). The pH increased in both ABC and WBC treatments, and also in the case of ABCxAD treatments. Similar increases in the salt content were detected in ABC-treated samples and in ABCxAD treatments at higher application rates. WBC increased the water holding capacity and carbon content of the soil. Phytotoxic effects of biochars were not detected, although higher doses resulted in slower germination. Combined biochar–digestate applications resulted in increased plant yields compared to sole biochar treatments. Thus, biochar–digestate combinations appear to be applicable as organo-mineral fertilizers.

1. Introduction

Biochar is a carbon-rich product of the pyrolysis process intended to be used as a soil amendment to improve the physical and chemical characteristics of soil and mitigate climate change by sequestering carbon. When applied to soil, biochars, due to their large internal surface area, increase soil porosity, cation exchange capacity (CEC), and water holding capacity (WHC), improve the soil as a microbial habitat, and provide plant nutrients [1,2,3,4,5,6,7,8,9]. Biochar is a thermochemically modified product with high carbon content, large specific surface porous structure, and various surface functional groups formed during the pyrolysis of biomass under anoxic conditions at 450–550 °C (plant biomass) or 550–850 °C (animal by-products). It can be obtained from a wide range of feed materials, and the product is highly varied in composition in relation to both origin and manufacturing process [10]. Besides its agricultural benefits, biochar applications are suitable for reducing organic waste [11], the emissions of greenhouse gases [12,13,14,15,16], and nitrate leaching [15,17]. The application of biochars can help to protect water resources by adsorbing potential contaminants [18,19,20,21,22] and by reducing nutrient leaching [2]. Therefore, the use of biochar as a soil amendment is an innovative and highly promising practice for sustainable agriculture [14,23,24].

Several greenhouse and field studies have been performed to examine the effects of biochars on soil properties and crop yields [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44]. Most studies show that the application of biochar increases crop yield, but no difference, or even negative results, have also been reported in a few cases [33,34,35,36,37,38,39,40,41]. The various effects on crop yield appear to depend on factors such as the quality of biochar (based on the type of feedstock and the condition of pyrolysis), the amount of applied biochar, soil type, and the tested crop; thus, the harmonization and adaptation of archive and new data and further new assessments are needed to understand the interactions among various biochars, soils, climate, and crops [23].

The pH of biochars is usually neutral to basic; thus, biochar application has a liming effect on soils, which plays an essential role in increasing plant productivity [11]. Yet, biochar can also produce phytotoxic effects due to extreme levels of soluble salts and its high pH [45]. Soluble salts are often present in the ash fraction of biochars, mostly depending on the mineral content of the feedstock [11]. Biochar made from poultry litter, can contain high levels of soluble salts, whereas plant-derived biochars usually show smaller ratios of inorganic components [46].

Numerous studies have confirmed that biochars of various origin are very efficient absorbents for nutrients [1,47], such as dissolved ammonium [17,48,49,50] and phosphate [2,51], although the increase in phosphorus pool in the soil as a result of biochar amendments has been linked to primary and occluded phosphorus, and only at a negligible rate to organic phosphorus content [52]. The absorbance and slow release of plant nutrients of such biochar varieties provide prospects to reduce the amount of fertilizers used in agricultural production systems. The efficacy of biochar application on soil characteristics and crop yield has been assessed not only for the individual application of biochar, but also for combined applications of biochar in or with compost [53,54], or with chemical fertilizers [55]. The results indicate that the combined application of biochar with chemical fertilizers can lead to substantial promoting effects on crop yield, but unchanged improvements on soil organic carbon (OC) content and reduced global warming potential.

Anaerobic digestion (AD) is a microbiological process that transforms biodegradable materials into their metabolites resulting in different beneficial products [56], such as biogas (energy) and nutrient-rich solid or liquid by-product (digestate) that can be used as a potential fertilizer in agriculture [57,58,59]. A wide range of organic substances can be used as starter materials in AD factories. The raw material or substrate of the AD process can contain different animal manures and slurries, energy crops, solid field residues [60], and animal by-products, as well as food waste, organic fractions of municipal solid waste, sewage sludge, etc. [61,62,63,64,65,66]. During digestion, dry matter is partially degraded depending on its chemical composition [67]. Digestate has high NH₄⁺-N content [60,68], decreased organic dry matter and total carbon content, elevated pH value [69], and a small C/N ratio [60]; additionally, the total level of nutrients remains nearly unchanged during the process [70]. In addition to its nutritive effect, digestate as a significantly complex material affects a wide range of physical, chemical, and biological properties of soil, depending on soil types [71]. The nitrogen content of digestate is a particularly important characteristic, as nitrogen is commonly considered to be the key factor limiting crop growth in agriculture [72,73]. The high NH₄⁺-N content of the digestate increases the nitrogen content of soils; however, it is easily oxidized to nitrate [74]. Similar to the alteration of nitrogen content, the application of digestate results in a possible increase in the bioavailable potassium content, but any significant fluctuation in the bioavailable phosphorus content of the soil has not been detected [75]. The application of digestates on land without proper precaution may have disadvantages as well. Due to the use of biogas, residue changes in the pH can be detected [76,77]. In addition, the use of liquid digestates may lead to NH₃ volatilization and nutrient loss; moreover, the eutrophication of nearby water systems can be caused by leaching [78]. Both amendments seem to influence each other’s effect, but their interaction is not well studied [79]. Based on the positive combined effects observed in the co-application of biochar with chemical fertilizers [55], it appears achievable to gain mutual benefits from the combined application of biochar with anaerobic digestate. Thus, the co-application of biochar and digestate lead to improvements in several soil quality properties, but have a negative effect on soil respiration [80]. As for the beneficial effects on crops, the combined use of biochar with liquid digestate has been found to be favorable in increasing fruit yield (fruit number and weight) and quality (sugar content and consistency) in organic tomato production [81]. It has also elevated the biomass production of lettuce [80], and treatments with combinations of wood-based biochars and liquid or solid fractions of anaerobic digestates achieved yields comparable to the treatment using a commercial fertilizer also on lettuce and two other vegetables (kale and rocket salad) [59]. In addition, the combined application of biochar and anaerobic digestate resulted in improved crop performances (number of leaves and total branches, plant dry weight) and the final essential oil content in the cultivated aromatic plant rose-scented geranium [82].

Therefore, the objective of this study was to investigate the effects of two types of biochar and the combined application of biochar and digestate on selected physical and chemical soil properties. In addition, the effects of treatments (different biochars and biochar–digestate combinations) on the yield of the ryegrass (Lolium perenne) and the uptake of certain elements were also determined.

2. Materials and Methods

2.1. Soil Sampling and Soil Properties

Soil samples were collected from the Ap horizon (0–30 cm) of an Arenosol obtained from a research field at the Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary. The soil samples were stored under cool (+5 °C) and dry conditions until the measurements. The air-dried samples were homogenized and passed through a 2 mm sieve prior to the analysis. Soil texture was characterized by relative proportions of sand (2–0.02 mm), silt (0.02–0.002 mm), and clay (<0.002 mm) content of 78%, 9%, and 13%, respectively (sandy loam). The organic matter content (0.49%) and, within that, the hydrophilic dissolved OC content (0.08%), as well as the total salt content (0.028%) were very low, and the top horizon was generally acidic (pH_H2O− 5.5; pH_KCl 4.2). In every case, the AL (ammonium acetate lactate) method was used as a Hungarian Standard to measure the plant’s available P and K. The ammonium–lactate–acetate soluble phosphate (AL-P₂O₅) and potassium (AL-K₂O) content of the model soil (111.1 mg kg⁻¹ and 119.9 mg kg⁻¹, respectively) were rather low according to the Hungarian limits. The results of the physical and chemical analyses showed that the CEC value was characteristically low (12.2 cmol kg⁻¹) for sandy loam soil, and the WHC was also relatively low (21.5%).

2.2. Biochars

Wood chip biochar (WBC) and animal bone biochar (ABC) were produced by Terra Humana Ltd. (Polgárdi, Hungary) with the use of 3R zero-emission pyrolysis technology. Biochar samples were transferred and stored at room temperature. Biochar properties, measured by Wessling Hungary Ltd. (Budapest, Hungary), are summarized in

Table 1. Characteristics of the two applied biochars.

Parameter	WBC *	ABC *	Parameter	WBC *	ABC *
Bulk density (g cm−3)	0.36	0.31	Potassium (mg kg−1)	4450	2000
Dry matter (%)	93.87	99.95	Potassium (AL) (mg kg−1)	1450	1500
Ignition residue (ash) of dry matter (%)	11.61	100	Magnesium (mg kg−1)	1200	6000
Total carbon (%)	79.8	9.9	Manganese (mg kg−1)	1140	1
Total nitrogen (%)	0.7	1.8	Sodium (mg kg−1)	170	7000
C/N ratio (%)	99.4	5.1	Phosphorus (mg kg−1)	780	133,000
pH	8.32	7.58	Phosphorus (AL) (mg kg−1)	214	24,600
CEC (cmol kg−1)	14.7	n.d. **	Zinc (mg kg−1)	41	152
Calcium (mg kg−1)	30,200	300,000	Sum of PAH (mg kg−1) ***	4.82	0.37
Chromium (mg kg−1)	4	4	Sum of PCB ***	-	-
Copper (mg kg−1)	9	5	Nitrite (KCl) (mg kg−1)	0.4	0.6
Iron (mg kg−1)	2280	63	Nitrate (KCl) (mg kg−1)	<10	<10

* WBC: wood chip biochar; ABC: animal bone biochar; ** n.d.: not determined; *** PAH: polycyclic aromatic hydrocarbons; PCB: polychlorinated biphenyls.

.

2.3. Liquid Digestate

Liquid digestate was obtained from the output of the fourth largest AD factory in Hungary using 3R zero-emission pyrolysis and phosphorus recovery technology with four digesters (2 × 2400, 2 × 1200 m³) and an industrial-scale pyrolysis plant installation/operation permit. Its overall throughput capacity was over 20,800 tons per year. The process was based on a horizontally arranged, indirectly heated rotary kiln providing the reductive thermal decomposition of any biomass at material core temperatures as high as 850 °C in vacuum. Plants were treated with different agricultural (whey, cow slurry, cow manure), food industrial by-products (treated slaughterhouse waste, treated food waste, grease trap waste, canola, sunflower oil production waste, acetic acid), and sewage sludge. The digester feeding system operated in a one-step continuous mode, and the plant operated under wet conditions at mesophilic temperatures (40–41 °C). The substrate was fed into the two larger digesters at volume rates of approximately 1.5–1.6 m³ per hour. The hydraulic retention time was approximately 60 days. The samples were stored at a low temperature (+5 °C) until use. Liquid digestate properties are summarized in

Table 2. The basic chemical properties of the applied digestate.

Parameter	SSD *	Parameter	SSD *
pH	7.51	Cr (VI) (mg kg−1)	1.03
Density (g cm−3)	0.995	Cu (mg kg−1)	166.9
Dry matter (m m−1 %)	5.04	Fe (mg kg−1)	10,853
Organic matter (m m−1 %)	3.28	Hg (mg kg−1)	0.12
Total N (mg kg−1)	104,683	Mg (mg kg−1)	8948
Total P (mg kg−1)	28,175	Mn (mg kg−1)	291.7
Total K (mg kg−1)	13,036	Mo (mg kg−1)	4.62
As (mg kg−1)	3.43	Na (mg kg−1)	11,706
Ca (mg kg−1)	57,937	Ni (mg kg−1)	19.5
Cd (mg kg−1)	0.69	Pb (mg kg−1)	20.0
Co (mg kg−1)	8.23	Se (mg kg−1)	3.51
Total Cr (mg kg−1)	21.2	Zn (mg kg−1)	748.0

* SSD: Sewage sludge digestate.

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2.4. Experimental Design

The experiment performed with pot-grown plants was carried out under laboratory conditions using plastic containers. Amounts of biochars corresponding to preset mass ratios were added to the soil. Biochar applications to the soil were conducted at four levels, 1% (10 g kg⁻¹), 2.5% (25 g kg⁻¹), 5% (50 g kg⁻¹), and 10% (100 g kg⁻¹). Each 1000 g biochar-soil mixture sample was watered with 150 cm³ kg⁻¹ deionized water or deionized water with liquid digestate (170 kg ha⁻¹ equivalent N). The amount applied corresponded to 60% of the soil water holding capacity. These mixtures were divided into four plastic containers with a closed bottom to avoid the loss of water and nutrients. Each container was filled with 200 g of biochar-amended soils. The remaining soil was used in other experiments. Lolium perenne seeds (2 g) were sown into each pot. This type of test was performed as a bio-indicator of phytotoxicity [83,84]. During the test, the moisture content of pots was monitored by daily weighing and the irrigation of the pots according to their weight. After 4 weeks, the germinated plants were harvested from each pot. Samples were dried at room temperature (25 °C) before the measurements.

2.5. Soil and Plant Analysis

Soil samples from each pot were passed through a 2 mm sieve to separate the plant roots and debris. Samples were analyzed according to the relevant Hungarian Standards. The total water-soluble salt content was measured by determining electrical conductivity (EC) (1:10 mass ratio) using a Radelkis OK-114 conductometer (Radelkis Ltd., Budapest, Hungary). The pH (H₂O) from the 1:2.5 mass ratio was detected using a Radelkis OP-211/3 laboratory pH meter (Radelkis Ltd., Budapest, Hungary). AL-soluble P₂O₅ was measured using a Spekol 221 UV-VIS spectrophotometer (C. Zeiss, Jena, Germany). K₂O was measured using a Jenway PFP7 flame photometer (Cole-Palmer Ltd., Staffordshire, UK). The WHC was determined by the modified funnel method by Bernard [85]. The OC content was assessed by the standard operating procedure of the Food and Agriculture Organization (FAO) of the United Nations based on the Tyurin method [86] using a Perkin Elmer 3100 atomic absorption spectrometer (PerkinElmer, Inc., Waltham, MA, USA) on the samples digested by cc. HNO₃ + H₂O₂ (8:2), and subsequently diluted with H₂O. Measured parameters from the plant samples were: total P, total K, and micronutrients (Ca, Mg, Cu, Fe, Mn, Zn, and Cr). During the cultivation period, growth rates were evaluated based on average grass height; the heights of 25 individual blades of the germinated plants were measured using a digital caliper, and their average was calculated daily.

2.6. Statistical Analysis

Differences in soil properties between the treatments (at four application rates plus control for each biochar and combination type) were analyzed statistically using the statistical program R 4.0 (The R Foundation for Statistical Computing, Vienna, Austria). The effects of the factors (biochar type, application rate, and the presence of anaerobic digestate), as well as their interactions with the physical and chemical characteristics of the soil samples, were investigated; moreover, the yield (air-dry mass) of ryegrass (Lolium perenne) and the uptake of certain elements were analyzed with the use of three-way analysis of variance (ANOVA). The main effects on plant yield were assessed using general linear models based on four experimental factors considered (biochar type, application rate, the presence of digestate, and time). The applicability of the fitted model was checked in each case with diagnostic plots (residual variances, diagnostic plots (QQ plot), Cook’s distance plot). In cases where a three-way interaction was identified among the factors investigated, the three-way ANOVA analysis was divided into two-way ANOVA analysis to interpret the main effects and interactions. Prior to the statistical analyses performed in R 4.0, the normality of the data and the homogeneity of variance were checked in each case by a Q–Q plot using Shapiro–Wilk and Levene’s or Bartlett’s tests. In cases where the conditions for the application of the chosen statistical method were not met, log transformation of the data was performed. Tukey’s honest significant difference (HSD) tests were conducted as post hoc analyses to assess the significant differences between groups.

3. Results

3.1. Soil Physical and Chemical Characteristics

Based on the results of the statistical analysis, all of the investigated factors (biochar type, biochar rate, and the presence of digestate) significantly affected the concentration of AL-P₂O₅ (p < 0.001), and a statistically significant three-way interaction was proven among biochar type, biochar rate, and the presence of digestate (p < 0.001). During further analysis, the significant effects of parameters and their interactions were detected, with one exception, where the individual effect of the presence of digestate was not significant on the level of AL-P₂O₅ at the lowest application rate (1%) (p = 0.672) (

Table 3. Effects and interactions of the investigated factors on soil physical and chemical characteristics in the investigated soil samples.

Investigated Factor/Parameter	AL-P2O5	AL-K2O	pH	Salt	Carbon	WHC
	p Values *
Type:Rate:Digestate	3.55 × 10−7	1.77 × 10−4	1.15 × 10−5	0.002	0.124	0.003
Type:Rate	<2 × 10−16	0.001	2.10 × 10−10	1.27 × 10−11	0.001	<2 × 10−16
Biochar type	<2 × 10−16	2.33 × 10−7	<2 × 10−16	1.15 × 10−6	6.25 × 10−5	<2 × 10−16
Biochar rate	5.21 × 10−16	6.00 × 10−9	8.43 × 10−16	0.002	9.39 × 10−4	<2 × 10−16
Type:Rate (xAD)	7.47 × 10−13	3.68 × 10−5	8.41 × 10−16	0.002	5.01 × 10−4	<2 × 10−16
Biochar type	5.00 × 10−16	3.51 × 10−9	<2 × 10−16	3.56 × 10−5	7.95 × 10−5	<2 × 10−16
Biochar rate	8.03 × 10−13	7.72 × 10−11	3.76 × 10−15	0.002	5.40 × 10−4	<2 × 10−16
Type:Digestate (1%)	0.017	0.091	4.77 × 10−8	0.901	0.355	8.15 × 10−4
Biochar type	9.43 × 10−8	0.004	2.07 × 10−7	2.81 × 10−4	0.601	0.071
Digestate	0.672	0.158	1.59 × 10−6	0.003	0.216	0.002
Type:Digestate (2.5%)	3.32 × 10−7	2.79 × 10−4	4.06 × 10−4	0.003	0.540	0.005
Biochar type	4.57 × 10−10	2.79 × 10−4	2.47 × 10−6	0.006	0.191	2.15 × 10−7
Digestate	4.94 × 10−7	0.257	1.83 × 10−4	0.026	0.716	0.332
Type:Digestate (5%)	6.05 × 10−5	9.19 × 10−4	0.006	0.011	0.237	0.823
Biochar type	8.55 × 10−10	0.003	1.36 × 10−9	3.30 × 10−5	7.26 × 10−7	3.11 × 10−8
Digestate	1.64 × 10−4	0.895	0.437	0.299	0.565	0.027
Type:Digestate (10%)	1.26 × 10−9	1.52 × 10−5	0.043	0.572	0.169	0.038
Biochar type	3.97 × 10−4	0.027	2.78 × 10−11	3.46 × 10−6	0.006	4.47 × 10−12
Digestate	2.67 × 10−4	0.023	0.036	0.059	0.267	0.745
Rate:Digestate (WBC)	8.02 × 10−5	0.002	3.76 × 10−7	0.180	0.188	0.021
Biochar rate	1.44 × 10−4	3.70 × 10−10	2.15 × 10−5	0.180	3.01 × 10−5	<2 × 10−16
Digestate	5.04 × 10−13	0.008	2.36 × 10−9	2.19 × 10−4	0.476	0.098
Rate:Digestate (ABC)	4.12 × 10−5	1.01 × 10−7	0.030	1.96 × 10−7	0.900	0.005
Biochar rate	2.75 × 10−15	0.013	<2 × 10−16	1.92 × 10−14	0.726	7.25 × 10−8
Digestate	2.06 × 10−9	0.037	5.83 × 10−4	0.243	0.729	0.412

* Statistical probabilities of obtaining the observed results, assuming that the null hypothesis was not true; p values are indicated in bold if not exceeding the value of 0.05 (95% probability that the result was conclusive).

). Based on group comparisons, the concentration of AL-P₂O₅ was higher (413.7 ± 50.8 mg kg⁻¹) in the digestate-supplemented control samples (ControlxAD), in contrast to the control without the digestate (111.1 ± 1.0 mg kg⁻¹) (p = 0.009). Treatments were carried out with the two types of biochar without digestate (WBC and ABC) and in combination with anaerobic digestate (WBCxAD and ABCxAD). Compared to the control, the AL-P₂O₅ content of the WBC-treated soils significantly increased (p < 0.001); however, the difference was not significant at the lowest (1%) and highest application rates (10%) (p > 0.148). After the WBCxAD treatment, lower AL-P₂O₅ content was determined in the samples compared to the control measured with the digestate (ControlxAD) (p < 0.007). ABC (p < 0.010) and ABCxAD (p < 0.043) treatments resulted in significantly higher concentrations of AL-P₂O₅ compared to the corresponding control groups. The presence of digestate resulted in higher concentrations in the case of WBC (p < 0.001), while significantly lower concentrations were measured for ABC from the application rate of 2.5% toward the highest rate (p < 0.004). The AL-P₂O₅ concentration was higher in samples treated with ABC than for WBC treatment without the digestate (p < 0.001). In samples treated with ABCxAD, the concentrations were also higher compared to WBCxAD; however, the increased content was only statistically significant for application rates of 1% and 2.5% (p < 0.001). The increasing easily soluble phosphorus content correlated well (R² = 0.982) with the increasing ABC application rates. In ABCxAD treatments, the same trends were observed (R² = 0.997) (Figure 1A).

Similarly to AL-P₂O₅ content, a statistically significant three-way interaction was detected among the investigated factors (biochar type, biochar rate, and the presence of digestate) (p < 0.001). Based on the analysis, the effects of the application rates individually (p ≤ 0.013), as well as in interaction with the biochar type (p ≤ 0.001), and the presence of digestate (p ≤ 0.002) were significant as well. The presence of digestate affected the AL-P₂O₅ content significantly only at the highest application rate (p = 0.023), while at the lowest application rate, the concentration significantly depended on the type of the biochar (p = 0.004) (Table 3). In the ControlxAD samples, higher AL-K₂O content (133.9 ± 4.5 mg kg⁻¹) was detected, than in control samples without the digestate (119.9 ± 3.6 mg kg⁻¹) (p = 0.001). WBC and WBCxAD treatments increased the AL-K₂O content of the soil compared to the corresponding controls (p < 0.005), although the difference was not significant below application rates of 2.5% for WBCxAD treatments (p ≥ 0.429). ABC resulted in significantly higher AL-K₂O content at the higher application rates (5 and 10%) (p < 0.001), compared to the control. In ABCxAD-treated soil samples, lower AL-K₂O content was detected at lower application rates (1 and 2.5%) (p ≤ 0.006) compared to ControlxAD, while at the highest application rate (10%), a significantly higher concentration was observed (p = 0.004). In WBC-treated soil samples, significantly higher concentrations were observed compared to WBCxAD at the higher application rates (5% and 10%) (p < 0.037). A significant difference was detected between ABC and ABCxAD treatments at the application rate of 5% only (p = 0.026), where a higher level of AL-K₂O was measured for ABC. Generally, lower AL-K₂O concentrations were observed in soil samples treated with ABC than for WBC (p ≤ 0.005); however, the difference was not significant at the application rate of 5% (p = 0.562). Between application rates of 2.5 and 10%, the same tendency was observed; thus, significantly lower AL-K₂O content was detected for ABCxAD of animal origin compared to WBCxAD (p ≤ 0.004). Based on our measurements, the increase in AL-K₂O concentration correlated with the increasing application rates of WBC (R² = 0.951) and WBCxAD treatments (R² = 0.963) (Figure 1B).

A statistically significant three-way interaction between biochar type, application rate, and the presence of digestate was detected in measured pH values (p < 0.001). The significant effects of parameters and their interactions were detected as well, with only one exception, where the individual effect of the presence of digestate was not significant on the pH at the application rate of 5% (p = 0.437) (Table 3). Higher pH values were detected in ControlxAD (6.09 ± 0.02) than in the control without digestate (5.54 ± 0.04) (p < 0.001). WBC increased soil pH significantly at higher (5% and 10%) application rates only (p < 0.001), by 0.2–0.4 units compared to the control. ABC treatments increased soil pH—relative to the control—in all application rates (p < 0.001) by 0.5 to 2 units, with the highest increase observed for the highest ABC rate. WBCxAD resulted in significantly lower pH at the higher application rates of 5 and 10% (p ≤ 0.006) compared to ControlxAD. From the application rate of 2.5% to higher application rates, ABCxAD caused significantly higher pH in soil samples compared to the ControlxAD (p ≤ 0.001), but with lower intensity (by 0.2 to 1.3 units) than in treatments without digestate. Significantly higher pH was detected in the WBCxAD-treated samples, compared to samples treated with WBC (p < 0.039); however, at the highest application rate (10%), the difference was not significant (p = 0.900). In ABC-treated soil samples, significantly higher pH values were detected at the lowest (1%) application rate compared to the ABCxAD-treated soil samples (p = 0.004). Moreover, significantly higher pH values were observed in ABC-treated soil samples compared to the WBC treatment as well (p < 0.001). In the presence of digestate, between application rates of 2.5 and 10%, pH values were also significantly higher for ABCxAD (p < 0.001) than for WBCxAD. A good correlation was found between the pH values and application rates of WBC (R² = 0.915), ABC (R² = 0.989), and ABCxAD treatments (R² = 0.965) (Figure 1C).

In the case of the water-soluble salt content, a statistically significant three-way interaction was proven among the investigated factors (biochar type, application rate, and the presence of digestate) (p = 0.002). The salt content of the samples investigated was highly affected by biochar type and the application rates; moreover, their interaction was significant as well (p ≤ 0.0002). At the lowest application rate of 1%, salt content depended on the type of biochar and the presence of digestate (p ≤ 0.003), but the interaction of the two factors was not significant (p = 0.901). In contrast, at the higher application rates of 5 and 10%, the effect of the presence of digestate on the salt content was not indicated (p ≥ 0.059), and the combined effect of the presence of digestate with biochar type was not even significant for the 10% application rate (p = 0.572). In WBC-treated samples, the salt content was found to be significantly affected only by the presence of digestate (p < 0.001), while in ABC-treated samples, only the presence of digestate did not affect the salt content (p = 0.243); thus, the salt concentration was affected by the application rate and by the interaction of the presence of digestate and the application rate (p < 0.001). A significant difference in the water-soluble salt content of the control without digestate (0.028 ± 0.001%) and with the presence of digestate (ControlxAD: 0.037 ± 0.004%) was not detected (p = 0.184) based on the measurements, despite the notable difference between the average values of the two types of controls. Compared to the control, in soil samples treated with WBC, a slight but significant decrease in the salt content was observed at the lower application rates (1 and 2.5%) (p = 0.0.45). In contrast to WBC, ABC resulted in a linear increase in soluble salts with the application rates compared to the control (p < 0.001), but the salt content remained below 0.07% even at the highest (10%) application rate of the ABC treatment, and at the lowest application rate (1%) the difference was not statistically significant (p = 1.000). The same trend was found in ABCxAD treatments, but the increase in the salt content was statistically significant only at the higher application rates (5 and 10%) (p ≤ 0.003), while significant differences were not detected in WBCxAD-treated samples compared to ControlxAD (p ≥ 0.444). Samples treated with WBCxAD resulted in higher soluble salt contents than in samples treated with WBC only; however, the difference was significant only for the application rate of 2.5% (p = 0.022). In the case of ABC, significantly higher salt content was observed compared to ABCxAD-treated samples; however, the difference was not significant at the application rate of 2.5% (p = 0.116). Compared to WBC, in ABC-treated samples, significantly higher soluble salt contents were detected (p ≤ 0.002) in the digestate-supplemented treatments. Moreover, higher soluble salt contents were detected in the ABCxAD sample of animal origin than in the WBCxAD-treated samples, although at the application rate of 2.5% the difference was not significant (p ≤ 0.035). According to our measurements, increasing salt content correlated well with increasing application rates of ABC (R² = 0.907) and ABCxAD treatments (R² = 0.977) (Figure 1D).

According to our results, the OC content of the investigated soil samples depended mainly on the biochar type and the application rate (p < 0.001); moreover, the interaction of the two main effectors also significantly affected the OC content in the samples analyzed (p ≤ 0.001). The biochar type had a significant effect on the OC content at the higher application rates (5 and 10%) (p ≤ 0.006); furthermore, in WBC-treated samples, the level of OC content highly depended on the application rate (p < 0.001) (Table 3). A significant difference in the OC content of the control groups (Control: 0.498 ± 0.006%, ControlxAD: 0.501 ± 0.028%) was not detected (p = 0.840). One of the aims of biochar application is to sustainably sequester carbon in soils. In our study, WBC treatments generally resulted in higher OC content in treated soils compared to the control, but significant increases were detected only at higher application rates (5 and 10%) (p ≤ 0.020), while changes in OC content were not found at any ABC application rate (p ≥ 0.795) compared to the control (Figure 1E). In the digestate-supplemented treatments, the same trends were detected, while a significant increase in the OC content was observed only for WBCxAD treatments at application rates of 5 and 10% (p < 0.042), while ABCxAD did not result in a change in OC content either (p ≥ 0.994). In soil samples treated with WBCxAD, significantly higher OC content was detected only at the lowest application (1%) rate (p = 0.038), compared to the samples treated with WBC, while significant differences between ABC and ABCxAD treatments were not detected based on the measured data (p ≥ 0.374). At the higher application rates (5 and 10%), significantly lower OC contents were determined for ABC compared to WBC treatment in the absence of digestate (p ≤ 0.034). In the presence of digestate, a significant difference between WBCxAD- and ABCxAD-treated samples was observed only at the application rate of 5% (p < 0.001). A good correlation between the application rates and OC content of investigated soils was detected for the WBC treatment (R² = 0.889) (Figure 1E).

According to our measurements, a statistically significant three-way interaction among biochar type, application rate, and the presence of digestate was detected in the rate of WHC (p = 0.003). Based on the statistical analysis, the rate of WHC highly depended on the type of biochar and application rate; moreover, the interaction of biochar type and application rate had a significant effect on WHC rates as well (p < 0.001). In the WBC- and ABC-treated samples, WHC highly depended on the application rate and its interaction with the presence of digestate (p < 0.001). At the application rates of 2.5% and 10%, the individual effect of the presence of digestate was not proven on WHC (p ≥ 0.332), while biochar type and the interaction of the presence of digestate and biochar type had a significant effect on the measured WHC rates (p ≤ 0.038). In contrast, at the application rate of 5%, the interaction of the two factors was not significant (p = 0.823). At the lowest application rate (1%), WHC values were significantly affected only by the presence of digestate and its interaction with biochar type (≤ 0.002). A significant difference in the WHC of the control groups (control: 23.12 ± 0.48%, ControlxAD: 23.54 ± 0.11%) was not detected (p < 0.001). WHC increased with increasing WBC (R² = 0.990) and WBCxAD application rates (R² = 0.986), with the highest increase observed for the highest WBC rate (p < 0.001); however, the increase in WHC at the lowest application rate (1%) was not significant (p = 0.250) compared to the control. Except for the application rate of 2.5% (p = 0.941), WHC was significantly higher after the ABC-treatment as well (p ≤ 0.031), compared to the control. In the digestate-supplemented treatments, higher WBC rates were observed compared to ControlxAD (WBCxAD: p < 0.001; ABCxAD: p ≤ 0.009). In soil samples treated with WBCxAD, a significant difference was only detected at the lowest application rate (1%) compared to WBC treatment (p = 0.001). At the highest application rate (10%) of ABC, significantly lower WHC was detected, than for ABCxAD treatment (p = 0.006). In WBC-treated soil, higher WHC levels were detected between the application rates of 2.5 and 10% compared to ABC treatments (p < 0.001). The same tendency was detected in the case of digestate-supplemented treatment as well, so higher WHC was measured for WBCxAD than in the ABCxAD-treated samples in the application range of 2.5 and 10% (Figure 1F).

3.2. Plant Growth and Yields

According to the statistical analysis, the interactions of the factors investigated significantly affected plant growth as well. A statistically significant three-way interaction was proven between biochar type, application rate, and time in digestate-supplemented samples compared to samples without digestate (p ≤ 0.005); moreover, three-way interaction was observed among biochar type, time, and the presence of digestate as well, but only at the highest application rate of 10% (p < 0.001). From the 10th day, the interaction between biochar type, presence of digestate, and application rate significantly affected plant growth (p < 0.001), and in ABC-treated samples, a three-way interaction between application rate, time, and the presence of digestate was detected (p < 0.001). In the investigated samples, a significant effect of biochar type (p ≥ 0.723) or an interaction of biochar type and application rate were not proven (p ≥ 0.441), while the application rate had a significant effect on plant growth during the experimental period, and in the digestate-supplemented samples (p ≤ 0.005). Plant growth was highly affected by the presence of digestate as well during the entire period of the experiment (p < 0.001) at most application rates (p ≤ 0.022) except for the highest (10%) one (p = 0.723), where the interaction of the presence of digestate and the biochar type significantly affected plant growth (p = 0.025). In addition to the presence of digestate, plant growth on the fifth day, was found to highly depend on the biochar type (p = 0.034) as well. In ABC-treated samples, plant growth was significantly affected by the presence of digestate, application rate, and their interaction as well (p ≤ 0.040), while in WBC-treated samples, the presence of digestate was the main effector (p < 0.001). Based on our data measured on the selected experimental days, plant growth was significantly affected by the application rate, the presence of digestate, and their interaction as well (p ≤ 0.025); however, the interaction of the application rate and the presence of digestate was not significant on the fifth day (p = 0.480). Based on data obtained from investigations of the effects on plant physiology, the observed plant growth values were significantly higher in the digestate-supplemented ControlxAD (p < 0.004), but the difference was not significant only on the 10th day (p = 0.331). Over time, a continuous increase in plant growth was detected in both control groups (p < 0.001). The measured plant growth values determined on the selected test days according to the control and treated groups are shown in Figure 2. In general, the average growth was higher for WBC during the growing period compared to ABC, and plant growth decreased with the higher application rates. In groups treated with WBC or ABC without digestate, significant differences were detected on the 15th day, while higher growth was observed in the group treated with WBC compared to ABC-treated samples at the application rate of 5% (p = 0.037). In contrast, at the 10% application rate, higher growth was detected for ABC treatment (p = 0.015) than in the WBC-treated samples. In the WBCxAD- and ABCxAD-treated groups, significantly higher growth was observed for WBC treatment at the highest application rate (10%) on observation days (p < 0.001). Over time, a continuous increase in plant growth was observed in the treated groups regardless of the type of the applied biochar, the use of digestate, or the application rates (p ≤ 0.003). Generally, in the samples, the combined biochar–digestate applications resulted in plants with increased growth in both types of biochar (WBCxAD: p ≤ 0.030, ABCxAD: p ≤ 0.041); however, the differences obtained were not significant in four cases of treatments with WBCxAD (5th day: 5% (p = 0.522); 10th day: 1% (p = 0.055) and 10% (p = 0.070); 20th day: 10% (p = 0.091) at various significance levels). In ABCxAD-treated samples, significantly higher growth was detected compared to ABC treatment (p < 0.023), except for the highest application rate (10%) of ABC, where the absence of digestate resulted in significantly lower growth on the 10th (p = 0.012), 15th (p = 0.001) and 20th (p = 0.001) days compared to ABCxAD. The observed higher growth in samples treated with the combination of biochar and the presence of digestate was most likely because plants could take up more nutrients from the soil due to the fertilization effect of the digestate (Figure 2).

A significant three-way interaction was detected between the investigated factors (biochar type, application rate, and the presence of digestate) based on the statistical analysis of air-dry mass values (p < 0.001). There were individual effects of the presence of digestate and the application rate, and their interaction was observed in both WBC- and ABC-treated samples (p ≤ 0.009). In the digestate-supplemented samples, the type of biochar and the application rate significantly affected the air-dry mass values, and an interaction of the factors was detected, while in samples without the digestate, air-dry mass was only affected by the application rate (p < 0.001). The air-dry mass values significantly depended on the presence of digestate at all application rates (p ≤ 0.014), while the biochar type had no effect (p ≥ 0.058), and the interaction of the biochar type and the presence of digestate was proven only at application rates of 2.5 and 10% (p ≤ 0.014) (

Table 4. Effects and interactions of the investigated factors on the yield (air-dry mass) of ryegrass (Lolium perenne) and uptake of certain elements at the end of the growing period based on plant tissue analysis.

Investigated Factor/Parameter	Air-Dry Mass	P Uptake	K Uptake	Ca Uptake	Mg Uptake	Mn Uptake
	p Value *
Type:Rate:Digestate	5.94 × 10−5	0.299	2.09 × 10−6	0.005	0.011	0.006
Type:Rate	0.579	0.091	1.73 × 10−6	2.49 × 10−6	3.46 × 10−7	3.96 × 10−8
Biochar type	0.334	0.004	2.64 × 10−7	1.60 × 10−8	4.11 × 10−10	8.67 × 10−16
Biochar rate	1.11 × 10−4	0.460	0.002	1.65 × 10−8	2.56 × 10−8	4.87 × 10−5
Type:Rate (xAD)	1.22 × 10−4	0.020	1.51 × 10−5	4.05 × 10−5	0.203	6.27 × 10−8
Biochar type	2.38 × 10−4	1.74 × 10−8	1.24 × 10−4	6.04 × 10−8	1.01 × 10−14	<2 × 10−16
Biochar rate	1.44 × 10−3	1.57 × 10−4	4.42 × 10−5	6.77 × 10−12	2.16 × 10−8	4.47 × 10−7
Type:Digestate (1%)	0.150	0.586	6.11 × 10−4	0.001	4.38 × 10−7	0.058
Biochar type	0.251	0.970	0.005	0.001	1.58 × 10−6	6.95 × 10−6
Digestate	4.55 × 10−5	8.19 × 10−6	0.096	6.78 × 10−8	7.54 × 10−9	0.002
Type:Digestate (2.5%)	6.95 × 10−3	0.173	0.493	0.261	3.47 × 10−4	0.093
Biochar type	0.058	0.418	0.086	0.016	3.55 × 10−7	2.27 × 10−10
Digestate	0.002	4.60 × 10−5	0.030	8.74 × 10−8	9.11 × 10−8	3.56 × 10−6
Type:Digestate (5%)	0.694	0.021	1.60 × 10−4	0.024	6.90 × 10−4	0.015
Biochar type	0.694	0.002	6.88 × 10−5	2.31 × 10−4	4.96 x10−6	8.48 × 10−9
Digestate	5.45 × 10−5	3.12 × 10−6	0.177	1.46 × 10−6	1.19 × 10−5	2.81 × 10−5
Type:Digestate (10%)	0.014	0.992	0.007	0.245	5.30 × 10−5	0.289
Biochar type	0.058	0.086	4.22 × 10−4	9.76 × 10−4	3.63 × 10−7	2.74 × 10−9
Digestate	0.014	0.002	0.024	0.339	0.028	5.01 × 10−4
Rate:Digestate (WBC)	0.001	0.823	0.048	4.92 × 10−8	8.70 × 10−7	6.98 × 10−7
Biochar rate	0.001	0.157	0.290	6.41 × 10−8	9.03 × 10−8	2.12 × 10−10
Digestate	1.95 × 10−8	1.17 × 10−6	0.207	6.23 × 10−10	2.62 × 10−6	3.57 × 10−6
Rate:Digestate (ABC)	0.009	9.94 × 10−4	1.54 × 10−7	6.24 × 10−5	5.12 × 10−7	1.72 × 10−6
Biochar rate	3.74 × 10−4	2.32 × 10−4	6.08 × 10−10	9.17 × 10−11	0.007	9.16 × 10−7
Digestate	3.72 × 10−6	1.03 × 10−11	0.018	8.27 × 10−9	3.98 × 10−15	0.002

* Statistical probabilities of obtaining the observed results, assuming that the null hypothesis was not true; p values are indicated in bold if not exceeding the value of 0.05 (95% probability that the result was conclusive).

). Significant differences in the yield of Lolium perenne of the control groups without (0.268 ± 0.013 g) and with the presence of digestate (ControlxAD: 0.288 ± 0.026 g) were not detected (p = 0.270). Based on our results, WBC treatments resulted in a significant increase in the plant yield only at the application rate of 2.5% (p ≤ 0.005) compared to the control, while between the ABC-treated and the ControlxAD groups significant differences were not detected at any application rates (p ≥ 0.095). WBCxAD resulted in higher plant yield compared to the ControlxAD value (p < 0.001); however, the difference was significant only at the application rate of 2.5% (p = 0.001). The presence of digestate resulted in higher plant yield in samples treated with WBCxAD (p ≤ 0.002) compared to WBC treatment; however, the increase in plant yield was not significant at the application rate of 2.5% (p = 0.492). The same trend (i.e., yield increase) was observed for ABC treatments (p ≤ 0.009), except for the highest application rate (10%), where in the presence and absence of digestate the difference was not significant (p = 1.000). Based on the measured plant yields, significant differences were not detected in samples treated with WBC and ABC (p ≥ 0.275), while in the presence of digestate significantly higher and significantly lower plant yield was observed for ABCxAD compared to WBCxAD at application rates of 2.5 (p = 0.005) and 10% (p = 0.044), respectively. Correlations between the application rates and plant yield were not detected in any of the treatments (R² ≤ 0.561) (Figure 3A).

3.3. Plant Tissue Analyses

Based on the statistical analysis of phosphorus uptake, significant three-way interaction was not detected among the investigated factors (biochar type, application rate, and the presence of digestate) (p = 0.299). In ABC-treated samples, phosphorus uptake was significantly affected by the presence of digestate and application rates individually, and their interaction was proven as well (p < 0.001), while in WBC-treated samples, phosphorus uptake was affected only by the presence of digestate (p < 0.001). In digestate-supplemented samples, significant effects of biochar type and application rate were found; moreover, their interaction was observed (p ≤ 0.020). In contrast to the digestate-supplemented samples, the only effective factor on phosphorus uptake was biochar type in the samples without digestate (p = 0.004). At the application rate of 5%, the biochar type and the presence of digestate, as well as their interaction, significantly affected the phosphorus uptake in the investigated plants (p ≤ 0.021), while at the other application rates, only the presence of digestate exerted a significant effect on phosphorus uptake (p ≤ 0.002) (Table 4). ABC contains high amounts of phosphorus (Table 1); thus, it may provide a good solution for the increasing global demand for phosphorus supply in agriculture. In the digestate-supplemented ControlxAD group, a higher level of phosphorus (41.48 ± 2.78 mg pot⁻¹) was detected compared to the control without digestate (33.39 ± 2.03 mg pot⁻¹) (p = 0.003). In our experiment, WBC treatments did not increase the phosphorus uptake significantly compared to the control (p ≥ 0.498), while the phosphorus uptake was higher in ABC-treatments than in the control (p ≤ 0.044); however, the difference was not significant at the lowest application rate (1%) (p = 0.358). Maximum uptake was observed at the higher, 5%, and 10% ABC application rates. Compared to ControlxAD, the phosphorus uptake decreased in all digestate-supplemented treatments for WBCxAD (p ≤ 0.002) and ABCxAD (p ≤ 0.002) as well, although it was increased at higher application rates, but still stayed below the control level. In the presence of digestate, lower phosphorus uptake was detected in WBCxAD-treated samples (p ≤ 0.003) compared to the individual biochar treatment (WBC); however, the difference was not significant at the highest application rate (10%) (p = 0.061). The same tendency, i.e., lower phosphorus uptake, was observed for ABCxAD (p ≤ 0.013) compared to the ABC treatment. Significant differences between the two types of biochar were only detected at the application rate of 5%, where higher uptake was proven for ABC (p = 0.006) compared to WBC, while in the presence of digestate lower uptake was detected for ABCxAD at the application rate of 2.5% (p = 0.014) than for WBCxAD. Correlation between the application rates and phosphorus uptake of plants was detected for WBC treatment (R² = 0.889) (Figure 3B).

Statistically significant three-way interaction appeared between biochar type, application rate, and the presence of digestate based on our measurements (p < 0.001). In the investigated samples, the biochar type and the application rate, as well as their interaction, significantly affected the potassium uptake (p ≤ 0.002). Similarly to phosphorus uptake, in ABC-treated samples, potassium uptake was significantly affected by the presence of digestate, application rates, and their interaction as well (p ≤ 0.018), while in WBC-treated samples, potassium uptake only depended on the interaction of the presence of digestate and the application rates (p = 0.048). At the highest application rate (10%), potassium uptake was significantly affected by the presence of digestate and the type of biochar (p ≤ 0.024); moreover, their interaction was proven as well (p = 0.007). At the other application rates, the main effective factors did not appear to be consistent; thus, at application rates of 1% and 5%, biochar type and the interaction of biochar type and the presence of digestate had a significant effect (p ≤ 0.005), while at the application rate of 2.5%, potassium uptake was affected only by the presence of digestate (p = 0.030) based on the plant tissue analysis results (Table 4). Significant differences in the potassium uptake of the control groups (control: 85.43 ± 3.39 mg pot⁻¹ and ControlxAD: 73.24 ± 8.84 mg pot⁻¹) were not detected (p = 0.061). The potassium uptake decreased in all WBC treatments compared to the control (p ≤ 0.003). The application of ABC resulted in decreased potassium uptake as well (p ≤ 0.002); however, the decrease was not significant at the application rate of 5% (p = 0.620) compared to the control. In the ABC treatments, maximum potassium uptake was observed at the 5% application rate, above that, the uptake decreased. In the case of the digestate-supplemented wood chip biochar (WBCxAD) treatments, lower potassium uptake was detected compared to the ControlxAD (p < 0.050), but the difference was not significant at the application rate of 5% (p = 0.582). In samples treated with ABCxAD, lower potassium uptake was detected as well (p ≤ 0.003), but the difference was not significant at the application rate of 5% (p = 0.194). In the presence of digestate, significantly lower potassium uptake was detected in WBCxAD-treated samples at the lowest application rate (1%) compared to WBC treatment (p < 0.001), while in the ABCxAD-treated samples, lower and higher potassium uptake was observed in the presence of digestate at application rates of 5% (p < 0.001) and 10% (p < 0.001) than in the ABC-treated samples. At an application rate of 1%, significantly lower potassium uptake was detected for ABC compared to WBC (p = 0.002), while at the higher application rates (5 and 10%), higher uptake values were observed for ABC (p ≤ 0.013). In the presence of digestate, significant differences between WBCxAD and ABCxAD were observed at the highest application rate of 10% (p = 0.008). Correlations between the application rates and potassium uptake were detected for WBC (R² = 0.884) and ABCxAD treatments (R² = 0.829) (Figure 3C).

In the case of calcium concentrations, a statistically significant three-way interaction was proven among the investigated factors (biochar type, application rate, and the presence of digestate) (p = 0.005). During further statistical analysis, biochar type (p ≤ 0.016) and application rate (p < 0.001) significantly affected calcium levels in the investigated samples; moreover, the interaction of biochar type and application rate was significant as well (p < 0.001). The interaction of application rate and the presence of digestate was significant as well (p < 0.001), but the interaction of the presence of digestate and the biochar type was not significant at application rates of 2.5% and 10% (p ≥ 0.245). At the highest application rate (10%), the individual effect of the presence of digestate was not proven either (p = 339); thus, the calcium concentration depended just on the type of biochar (Table 4). Based on our results, in the digestate-supplemented ControlxAB, a higher level of calcium (2357.9 ± 87.7 mg kg⁻¹) was detected compared to the control without digestate (1839.7 ± 37.3 mg kg⁻¹) (p = 0.003). Generally, a decrease in calcium content was observed in the test plants in all treatments compared to the controls (WBC: p < 0.048, WBCxAD: p < 0.001, ABC: p < 0.029, ABCxAD: p < 0.012), although at the lowest rate the decreased calcium content was significant only for WBCxAD treatment; furthermore, in the WBC treatment (p < 0.001), the 10% application rate showed an increasing tendency in calcium content, although it still stayed below the control level. In the presence of digestate, significantly higher calcium content was observed in WBCxAD- and ABCxAD-treated samples as well (WBCxAD: p < 0.001, ABCxAD: p < 0.001), compared to the corresponding biochar treatments without digestate WBC and ABC treatments; however, at the highest application rate (10%) the difference was not significant for either type of biochar (WBC: p = 0.059, ABC: p = 0.901). In treated samples without digestate, significantly higher calcium content was detected for WBC treatment compared to ABC at the higher application rate (10%) (p = 0.003). In the presence of digestate, higher calcium content was observed in ABCxAD-treated samples at the lowest application rate (1%) (p ≤ 0.042) compared to WBCxAD, while at a higher application rate of 5%, a higher level belonged to WBCxAD-treated samples (p = 0.002). According to our measurements, decreasing calcium content correlated well with increasing application rates of ABC (R² = 0.993) and ABCxAD treatments (R² = 0.992); in addition, a good correlation was found between the calcium content and application rates of WBC (R² = 0.835) and WBCxAD treatments as well (R² = 0.871) (Figure 3D).

Based on measured magnesium concentrations, a statistically significant three-way interaction was proven between the investigated factors (biochar type, application rate, and the presence of digestate) (p = 0.011). The interaction of biochar type and the presence of digestate (p < 0.001), and the presence of digestate and application rates were significant (p < 0.001); moreover, the individual effects of the factors significantly affected the magnesium content of the plant tissue (p ≤ 0.028). The interaction of biochar type and application rate significantly affected the magnesium level only in samples without the presence of digestate (p < 0.001), while in the digestate-supplemented samples, only the individual effects of biochar type and the application rate were significantly effective on the magnesium content (p < 0.001) (Table 4). According to our measurements, in the digestate-supplemented ControlxAD (1061.4 ± 50.8 mg kg⁻¹), a higher level of magnesium was detected compared to the control without digestate (674.2 ± 12.3 mg kg⁻¹) (p = 0.003). In samples treated with WBC, a lower level of magnesium was observed compared to the control (p = 0.001); however, the difference was not significant in the lowest application rate of 1% (p = 0.383). The same tendency (i.e., lower magnesium concentration) was observed in samples treated with ABC at lower application rates (1 and 2.5%) (p ≤ 0.019), while at the highest application rate (10%), significantly higher content was detected compared to the control (p = 0.038). The digestate-supplemented WBCxAD treatment resulted in a significant decrease in the magnesium content of the plants compared to the ControlxAD (p < 0.001). At the higher application rates of 5% and 10%, the digestate-supplemented ABCxAD treatment resulted in significantly lower magnesium content as well compared to the ControlxAD (p < 0.001). In the presence of digestate, significantly higher magnesium content was detected for WBCxAD-treated samples at application rates of 1 and 2.5% (p ≤ 0.001), while the highest application rate (10%) resulted in higher magnesium levels in the WBC treatment without digestate (p = 0.014). For ABC and ABCxAD treatments, the presence of digestate resulted in higher magnesium content in the measured samples (p < 0.001). Significantly higher magnesium contents were detected for ABC treatment than in the WBC-treated samples (p < 0.005), although at the lowest application rate (1%), the difference was not significant (p = 0.132). Higher levels of magnesium were detected for ABCxAD treatment in the presence of digestate as well (p < 0.001) compared to WBCxAD-treatment. According to our measurements, decreasing magnesium content correlated well with increasing application rates of ABCxAD (R² = 0.906) (Figure 3E).

Based on manganese content, a statistically significant three-way interaction was proven among the investigated factors (biochar type, application rate, and the presence of digestate) as well (p = 0.006). According to further statistical analysis, the type of biochar (p < 0.001), the presence of digestate (p ≤ 0.002), and the application rate (p < 0.001) significantly affected the level of manganese in the investigated samples; moreover, the interactions of biochar type and application rate were significant as well (p < 0.001). The interaction of the application rate and the presence of digestate was significant as well (p < 0.001), while the interaction of the presence of digestate and biochar type was significant only at the application rate of 5% (p = 0.015) (Table 4). Significant differences in the manganese content of the control groups without digestate (196.2 ± 16.6 mg kg⁻¹) and with the presence of digestate (ControlxAD: 147.4 ± 4.9 mg kg⁻¹) were not detected (p = 0.154). WBC treatments decreased the manganese concentration of the test plants compared to the control (p < 0.001) with increasing application rates, while in the case of ABC treatments, a significant reduction in manganese content was observed only at the lowest application rate (1%) (p = 0.018) compared to the control. The digestate-supplemented WBCxAD treatment resulted in a decreased manganese content at all application rates compared to ControlxAD (p < 0.001). In the ABCxAD treatment, increased manganese concentrations were detected at application rates of 2.5% (p = 0.009) and 10% (p < 0.001) compared to ControlxAD. In the presence of digestate WBCxAD, lower manganese content was detected at the application range of 1% and 5% (p ≤ 0.020) compared to WBC, while at the highest concentration (10%), a higher content of manganese was detected in the presence of digestate (p = 0.005) for WBCxAD treatment. The same tendency was observed for ABCxAD-treatment, with the only exception being that the reduced level of manganese was not significant at the lowest application rate (1%) (p = 0.275) in ABC- and ABCxAD-treated samples. Significantly higher levels of manganese were observed in ABC- and ABCxAD-treated samples compared to WBC and WBCxAD treatments in the presence and absence of digestate as well (p ≤ 0.002). In addition, a good correlation was found between manganese content and the application rate of WBCxAD (R² = 0.936) and ABCxAD treatments (R² = 0.816) (Figure 3F).

4. Discussion

4.1. Soil Physical and Chemical Characteristics

Based on the statistical analysis, biochar type, application rate, and the presence of digestate were significant predictors of the soil physical and chemical characteristics. Except for the OC content of the soils, a significant three-way interaction was proven among the investigated factors, but the interaction of application rate and biochar type had a significant effect on the measured values. AL-P₂O₅ and AL-K₂O content, as well as pH values, were highly affected by the presence of digestate and the application rate; moreover, their interaction was significant as well. Furthermore, AL-P₂O₅ was significantly affected by biochar type and its interaction with the presence of digestate. On the OC content of the investigated soil samples, the presence of digestate had no significant effect. The rate of WHC was significantly affected by the type of biochar and application rate; moreover, the interaction of the biochar type and the application rate had a significant effect on the WHC rates (Table 3).

ABC contains a high amount of phosphorus; thus, it can be a good option to use it as a phosphorus fertilizer in agriculture. The AL-P₂O₅ content of soil increased significantly with the ABC application rates (p < 0.010). The combined utilization of ABC and digestate (ABCxAD treatment) resulted in lower easily soluble phosphorus content compared to the ABC treatment in our study (p < 0.004); however, at the lowest application rate of 1%, the difference was not significant (p = 1.562). The reason for this effect could be explained by the calcium content of the applied digestate, which resulted in an additional amount of hardly soluble calcium phosphate forms.

Biochar treatments (ABC, WBC, ABCxAD, WBCxAD) increased the AL-K₂O content of the soil with the increasing application rates, although a statistically significant increase was observed only at higher application rates (5 and 10%) compared to the control groups (p < 0.028), while ABCxAD had a negative effect on the potassium content of the soil at lower application rates of 1 and 2.5% (p ≤ 0.006). This result is in agreement with reported data in the scientific literature [45]. However, at the highest application rate (10%), increased AL-K₂O content in the soil was detected after the ABCxAD treatment as well (p < 0.005).

Biochars usually have an alkaline pH and a liming effect that is often explained by the high content of calcium and sodium carbonates and oxides presented in biochar. Based on our results, ABC was more effective (p < 0.001) in increasing soil pH than WBC, as WBC increased soil pH only at the higher application rates (5 and 10%) and with lower units (p < 0.001). The increasing soil pH was in good agreement with the literature data [87,88].

The soluble salt content of the soils increased only in the ABC treatments (ABC: 2.5–10% p < 0.001, ABCxAD: 5–10% p ≤ 0.003), but it remained below 0.1% even at the highest 10% ABC treatment; thus, it did not cause salinity in the treated soils. The increasing water-soluble salt content in the soil was in good agreement with the published data by Smider and Singh [45].

Although biochar application is being considered as an efficient tool to sequester carbon, in our study, only the 5% WBC application rates significantly increased the OC content of the treated soils (WBC: 5–10% p ≤ 0.020, WBCxAD: 5–10% p < 0.042). This is possibly due to the short experiment period that was not sufficient for the proper incorporation of biochars into the treated soils. We also suppose that the applied analytical method (Tyurin method) could indicate a higher error in the results because the oxidizing reagent (K₂Cr₂O₇) hardly oxidized the graphite layers of the biochar. The digestate-supplemented treatments improved the OC content of the soils significantly only at the higher application rates (5 and 10%) and only in samples treated with WBCxAD in this experiment. The organic fractions of digestate can contribute to soil organic matter, but its fertilizer effect is more impactful in contribution to soil organic matter turnover [72].

Due to high surface area and porous structure, WBC application resulted in a linearly increasing WHC of the treated soils (p < 0.001); however, at the lowest application rate, the increase was not significant (p = 0.250). ABC application significantly increased the WHC value too (p ≤ 0.031), except for the 2.5% application rate (p = 0.941), possibly due to its hydrophobic nature. Based on the literature data, the hydrophobic surfaces of biochars can be rapidly transformed in soil environments [89]; thus, field experiments are needed to investigate the long-term effects of ABC application on the WHC of soil.

4.2. Plant Growth and Yield

According to the statistical analysis, a significant three-way interaction was observed between application rate, biochar type, and time for the digestate-supplemented samples (p ≤ 0.005); furthermore, significance was observed between biochar type, time, and the presence of digestate at the highest application rate (p < 0.001). From the 10th day on, the interaction between the presence of digestate, the application rate, and biochar type significantly affected the plant growth. In ABC-treated samples, the three-way interaction between application rate, time, and presence of digestate was proven based on our data (p < 0.001). In the investigated samples, the application rate had a significant effect on plant growth in the digestate-supplemented samples (p ≤ 0.005), and plant growth was highly affected by the presence of digestate as well. Based on our data measured on the selected experimental days, plant growth was significantly affected by application rate, the presence of digestate, and their interaction as well, although the interaction of application rate and the presence of digestate was not found to be significant on the first sampling day (Table 4). Based on our results, generally higher average plant growth resulted from WBC treatments during the growing period compared to ABC, but the difference was primarily significant only at the higher application rates (p < 0.037). The high nutrient content of digestate resulted in higher nutrient rates in the plants (p < 0.040), but the combination of digestate and a higher dosage of biochars affected smaller plants in groups treated with ABC (p < 0.023) more than the other treatments. In another study [81], the highest values of total and marketable tomato yield were observed with a liquid digestate and pinewood biochar combination, where these results were related to the highest plant growth and fertility rates in terms of fruit number per plant, fruit weight, and aboveground biomass.

Based on air-dry mass values, a significant three-way interaction was detected between biochar type, application rate, and the presence of digestate. The significant individual effects of the presence of digestate and the application rate, as well as their interaction, were proven as well. In the digestate-supplemented samples, the type of biochar and the application rate significantly affected air-dry mass values, and the interaction of the factors was detected, while in samples without digestate, air-dry mass was only affected by the application rate (Table 4). The air-dry mass of the test plants (Lolium perenne) was significantly increased only in the digestate-supplemented WBCxAD and ABCxAD treatments (p ≤ 0.001); although, the difference was significant only at the application rate of 2.5% (p = 0.001) compared to the associated control. In general, the presence of digestate resulted in higher plant yield in samples treated with WBCxAD and ABCxAD compared to the WBC and ABC treatments. Our results, in agreement with similar data recently reported for lettuce (Lactuca sativa) [80], show that the digestate-supplemented ABC is more effective in the improvement of the air-dry mass of plants than digestate-free biochar treatments (p ≤ 0.009).

4.3. Plant Tissue Analyses

A significant three-way interaction was proven between application rate, biochar type, and the presence of digestate, based on the statistics, except for phosphorus uptake. In the digestate-supplemented samples, the presence of digestate, the biochar type, and the application rate, as well as the interaction of application rate and the presence of digestate, and the interaction of biochar type and application rate had significant effects on phosphorus uptake. In the investigated samples, biochar type, application rate, and the presence of digestate were all significant predictors of the uptake of potassium, calcium, magnesium, and manganese. Significant effects of the interactions of biochar type and application rate, as well as the interaction of application rate and the presence of digestate, were proven, as was the effect on the uptake of calcium, magnesium, and manganese. On the uptake of manganese, the interaction of the presence of digestate and biochar type had a significant effect for all of the application rates (Table 4).

Phosphorus is the second most important nutrient element (after nitrogen), and is limiting agricultural production in most regions of the world. Thus, phosphorus availability in different soil amendments is a very important factor in the planning of applications. In our study, a high phosphorus content of ABC-treated samples was observed between the application rates of 2.5 and 10% due to the increased phosphorus uptake of the plants (p ≤ 0.044), while the WBC did not have any effect on it (p ≥ 0.498) compared to the control. The combined biochar–digestate application treatments decreased the available P₂O₅ content, thus reducing the phosphorus uptake of the plants as well (p ≤ 0.002). We suppose that the high calcium content of the applied digestate resulted in hardly soluble calcium phosphate forms, and prevented the uptake of the phosphate by the plants.

In the case of the potassium, the test plants did not take up more K₂O than the control in all treatments (p < 0.029), except for the highest application rate (10%) of ABCxAD, which resulted in significantly higher K₂O content compared to all other treatments (p < 0.050).

The plants took up significantly less calcium in the ABC- (p < 0.030) and WBC-treated groups (p ≤ 0.012) than in the control groups. The biochar-free digestate application (ControlxAD) improved the calcium uptake of the plants (p = 0.003), but in combination with biochars (ABCxAD- and WBCxAD-treatments), significant decreases were observed (p < 0.001); however, in samples treated with ABCxAD, the decrease in calcium uptake was not yet significant (p = 0.059) at the lowest application rate of 1%, possibly due to the high carbonate content of the applied biochars that may form insoluble calcium carbonates under alkaline pH conditions.

The magnesium concentration of the test plants was affected significantly in the ABC and WBC treatments in our study, while significantly lower magnesium contents were observed in biochar-treated samples compared to the control (p ≤ 0.019); however, the difference was not significant at the lowest application rate of WBC treatment (p = 0.383) and the application rate of 5% in ABC-treated samples (p = 0.720), while at the highest application rate (10%), significantly higher content was detected in ABC-treated samples compared to the control (p = 0.038). Although digestate contains a high amount of magnesium, which is mainly in water-soluble form, the ABCxAD (application rates of 5% and 10%, p ≤ 0.010) and WBCxAD treatments (p < 0.001) caused decreasing magnesium content with increasing application rates in the plants, compared to the digestate-supplemented control treatment (ControlxAD), but magnesium concentration remained significantly higher than in ControlxAD and the biochar-free control.

In the case of manganese, ABC treatments affected the uptake only at the lowest application rate (1%) (p = 0.018) compared to the control, while the additional digestate applications (ABCxAD-treatments) significantly increased the manganese concentration of the test plants at application rates of 2.5 (p = 0.009) and 10% (p < 0.001) compared to the ControlxAD. The WBC treatments decreased the manganese concentration for all application rates (p < 0.001), and we found the same trend in all WBCxAD treatments as well (p < 0.001), except at the 10% application rate, where instead of a continuous decrease with the increase in the application rate, the growth in the manganese content was observed at the end of the experiment, compared to the value detected at the application rate of 5% (p < 0.001).

4.4. Limitations and Implications

The new EU Fertilizer Regulation (EC) No 2019/1009 will be officially implemented on 16 July 2022. This regulation expands the scope of fertilizers, not only including mineral and inorganic fertilizers, but also covering organic fertilizers, as well as biostimulants and fertilizers made from recycled substances. Due to this regulation, the market is open for these products. Although the materials used in our experiment are well known individually, their co-application has not been the center of attention, although the literature data indicate their mostly positive effects.

This study focuses on bioavailable nutrients, especially plant-available phosphorus. Numerous countries have developed recommendation strategies for phosphorus fertilizers. The determination protocols for the readily available phosphorus content in soils are different in various European countries. The AL (ammonium acetate lactate) method is the standard in Hungary and some other EU countries. Thus, the AL methods are used in the Hungarian Fertilizer Recommendation System to determine the easy soluble, plant-available phosphorus from the soil. That is why we measured these parameters from the materials and from the experimental samples, even if this method may overestimate the amount of plant-available phosphorus.

5. Conclusions

In conclusion, biochar type, application rate, the presence of digestate, and their proven interactions generally affected the investigated parameters in the soil and plant tissue samples, although not in consistent ways. We found that the applied ABC was a good source of available phosphorus as it increased the easily soluble P₂O₅ content of the treated soil. We suggest further research on the comparative analysis of biochars of various origins, as ABC may be a good option for contributing to phosphorus supply in the future. The pH and water-soluble salt content of the treated soil were increased significantly in the ABC treatments, while in the case of WBC treatments, the increase in pH was observed just at higher application rates, and significantly lower water-soluble salt content was detected at the lower application rates. The increase in pH and salt content did not cause very high alkalinity and/or salinity in either treatment. WBC increased WHC and OC content primarily at the higher application rates, while it reduced the potassium content of the treated soil. In contrast, no effects of ABC were observed on OC content. The application of ABC generally resulted in increased WHC and reduced potassium content as well.

As digestate can provide a high amount of valuable nutrients to plants, the combined application of biochar and digestate can be used as a replacement for mineral fertilizers. In our study, we found that the biochar–digestate combined treatments resulted in higher pH and P₂O₅ content of the soil in the ABCxAD-treated samples. In case of the WBCxAD treatments, lower potassium contents were observed in the soil. The co-application of these materials improved the reduction in environmental stress and the leaching of nutrients, and increased the amount of available nutrients for the plants. Though it seems a promising technique, further research is needed to provide new information and data on this topic, and to improve the effectiveness of the combined application of biochars and digestate in agricultural practice.