Digestate not only affects nutrient availability but also soil quality indicators

Citation: García-López, A.M.; Delgado, A.; Anjos, O.; Horta, C. Digestate Not Only Affects Nutrient Availability but Also Soil Quality Indicators. Agronomy 2023,13, 1308. https://doi.org/10.3390/ agronomy13051308 Academic Editor: Jiafa Luo Received: 30 March 2023 Revised: 27 April 2023 Accepted: 4 May 2023 Published: 6 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). agronomy Article Digestate Not Only Affects Nutrient Availability but Also Soil Quality Indicators Ana María García-López 1, Antonio Delgado 1, Ofélia Anjos 2,3,4 and Carmo Horta 2,5,* 1Department of Agronomy, ETSIA, University of Seville, Ctra. Utrera km 1, 41013 Seville, Spain; [email protected] (A.M.G.-L.); [email protected] (A.D.) 2Polytechnic Institute of Castelo Branco, School of Agriculture, Quinta da Sra. de Mércules, 6001-909 Castelo Branco, Portugal; [email protected] 3Spectroscopy and Chromatography Laboratory, Centro de Biotecnologia de Plantas da Beira Interior, 6001-909 Castelo Branco, Portugal 4Forest Research Centre, School of Agriculture, University of Lisbon and Associate Laboratory TERRA, 1349-017 Lisbon, Portugal 5CERNAS-IPCB Research Centre for Natural Resources, Environment and Society, Polytechnic Institute of Castelo Branco, 6001-909 Castelo Branco, Portugal *Correspondence: [email protected]; Tel.: +351-962950951; Fax: +351-272339901 Abstract: Digestate contains many essential nutrients for crops, including nitrogen (N) and phosphorus (P), and it can alter the biogeochemical cycle of nutrients and soil functionality. This work aimed to assess the fertilizing effects of digestate on chemical and biological soil properties in a field experiment in eastern Portugal with two horticultural crops involving nine treatments: control without fertilization; mineral N fertilization with 85 kg ha −1 ; fertilization with digestate (DG) with increasing N rates (85, 170, 255, or 340 kg N ha −1 ); and fertilization with different combinations of digestate plus mineral N (DG at 85 or 170 kg N plus 60 kg mineral N ha –1 or DG at 170 kg N plus 25 kg mineral N ha –1 ). In addition to N, digestate supplied significant amounts of P, Ca, K, and Mg and significantly increased soil Olsen P, mineral N, and organic C. At high doses, it decreased phosphatase and β -glucosidase activities, as well as fungi and bacterial biomass, compared to the control or mineral N fertilization, and it also negatively affected soil P and C cycling capacity and microbial biomass. The organic to total N ratio and the N to P ratio in digestate are crucial properties for evaluating its agronomic management as fertilizer. Keywords: phosphorus; nitrogen; enzymatic activity; organic amendment; soil microbial biomass 1. Introduction Facing the needs of a growing population requires increasing use of nitrogen (N) and phosphorus (P) fertilizers, particularly in developing countries, where crop yields are constrained by low fertilizer rates [ 1 ]. However, N and P can be considered nonrenewable resources; N fertilizer production relies on high energy consumption, and P fertilizer production depends on mine resources, whose production is expected to peak in the present century [ 2 , 3 ]. Agricultural production in many regions of the world, such as South America and Europe, is highly dependent on imported P [ 4 ]. Thus, N and P resources in agriculture may represent a major constraint on world food security in the near future. The P crisis in 2008 [ 5 , 6 ] and the fertilizer price crisis in 2022 are evidence of the volatility of the global fertilizer market and an expected price rise in the near future. Thus, reducing the amount of mineral N and P fertilizers applied while avoiding deficiencies in both nutrients is currently one of the main challenges in crop management to ensure more sustainable agricultural production and global food security [ 7 , 8 ]. Consequently, recycling any nutrient source to replace mineral fertilizers has become crucial for the sustainability of agricultural systems [ 9 ]. The by-product of the production of biogas with Agronomy 2023,13, 1308. https://doi.org/10.3390/agronomy13051308 https://www.mdpi.com/journal/agronomy Agronomy 2023,13, 1308 2 of 16 organic agricultural residues, the so-called anaerobic digestates, can be used as fertilizer [ 10 ]. However, adequate management of digestates is required to reduce negative agricultural and environmental impacts and to fully meet a circular economy approach. Currently, many studies have focused on the effects of digestates on crop and vegetable yields and quality [ 10 – 13 ]. Since digestates are usually used as fertilizers, they may contribute to the sustainable use of N and P resources in agriculture. However, the fertilizer value of digestates on soil is affected by the soil nutrient status, nutrient requirements of the crops, soil organic carbon (SOC), and the potential negative effects on crops due to phytotoxic compounds [ 14 ]. These potential negative effects can be decreased through composting, but this implies an additional cost [ 15 ]. Soil enzymatic activities are an important indicator of microbial activity, which is frequently altered by applications of fertilizers or organic amendments [ 16 , 17 ]. Digestate application contributes to increased soil organic C content. However, it may also promote the mineralization of native SOC through increased microbial activity ascribed to a “priming effect” [ 18 , 19 ]. This may have an impact on extracellular enzyme activities [ 19 – 23 ]. Increased hydrolytic enzyme activity resulting from organic amendments, such as phosphatases, may increase the mineralization of nutrients in organic form, enhancing nutrient cycling applied to soil as organic wastes or by-products [24]. In this context, to improve soil fertility and quality with digestate application, deeper knowledge about the effects of digestate on the availability and dynamics of nutrients and on the intrinsic nutrient cycling capacity of soil is needed. Previous research has shown that the combined use of organic and mineral fertilizers can maintain soil fertility while enhancing its functionality by increasing microbial biomass and activity [ 16 , 25 ]. This combined use of different nutrient sources contributes to a decrease in the use of non-renewable resources in agriculture. Therefore, the integrated management of nutrients and organic C by combining mineral and organic fertilizers is a recommended practice for more sustainable management of soil fertility [ 26 ]. In addition, the joint application of digestates with mineral fertilizer can reduce P adsorption and precipitation, thus increasing the recovery of applied P by crops [ 27 , 28 ]. Furthermore, this joint application of digestate and N fertilizer can, in the short term, help overcome the immobilization of N in the soil due to the temporary immobilization of N by soil microorganisms in treatments with digestates. All of this reveals the need for an integrated approach to the study of the effect of digestates on soil fertility and quality, with particular emphasis on their effects on N and P available pools and on the nutrient cycling capacity in soils. Thus, the present study investigated the effect of digestate application, alone or in combination with mineral fertilizers at different rates, on soil properties and functionality, such as soil pH, N and P availability, enzymatic activities, total microbial biomass (TMB), and microbial community. These effects were evaluated in a field experiment with two consecutive crops (lettuce and kale). 2. Materials and Methods The study was carried out in an experimental field of the Castelo Branco Polytechnic Institute of Castelo Branco, eastern Portugal (39.823655, − 7.451606). The field soil was an Inceptisol [ 29 ]. The main physiochemical properties are presented in Table 1. At the experimental site, average temperatures ranged from 7.1–24.6 ◦ C, with a mean annual value of 15 ◦ C. The annual precipitation was 735 mm, with very dry summers [ 30 ]. The climate conditions during both experiments are shown in Figure 1. Table 1. Soil properties. pH EC SOM C/N CEC Ca2+ Mg2+ Na2+ K+Total N Olsen P Pi Po dS m−1%cmol(+) kg−1g kg−1mg kg−1 6.4 0.10 5.4 8.3 36.9 7.30 0.90 0.02 1.37 2.43 149 663 373 ± sampling in February 2020. SOM, soil organic matter; CEC, cation exchange capacity; Pi, inorganic phosphorus; Po, organic phosphorus. Agronomy 2023,13, 1308 3 of 16 Agronomy 2023, 13, x FOR PEER REVIEW 3 of 17 Table 1. Soil properties. pH EC SOM C/N CEC Ca2+ Mg2+ Na2+ K + Total N Olsen P Pi Po dS m−1 % cmol(+) kg−1 g kg−1 mg kg−1 6.4 0.10 5.4 8.3 36.9 7.30 0.90 0.02 1.37 2.43 149 663 373 ± sampling in February 2020. SOM, soil organic matter; CEC, cation exchange capacity; Pi, inorganic phosphorus; Po, organic phosphorus. March-20 April-20 May-20 N.a.N. October-20 November-20 December-20 January21 February21 March-21 Rainfall (mm) 0 20 40 60 80 100 120 140 160 Temperature (ºC) 0 5 10 15 20 25 Rainfall Average temperature Lettuce Kale Figure 1. Climate conditions during the experiments. 2.1. Experimental Design An experiment with a fully randomized design and four replications was performed. Each replication corresponded to a plot of 1.08 m2 (1.20 × 0.9 m). The experiment was the same as reported by [31], and it involved nine treatments (Table 2): (i) no fertilization (control); (ii) mineral N fertilization at a rate of 85 kg ha−1 (Nm85) split in three applications (35 kg N ha−1 at preplant and two topdressings of 25 kg N ha−1); (iii) application of digestate (DG) equivalent to 85 kg N ha−1 (DG-N85); (iv) application of digestate equivalent to 170 kg N ha−1 (DG-N170); (v) application of digestate equivalent to 255 kg N ha−1 (DG-N255); (vi) application of digestate equivalent to 340 kg N ha−1 (DG-N340); (vii) application of digestate equivalent to 85 kg N ha−1 plus mineral nitrogen split in two applications (35 kg N ha−1 at preplant and 25 kg N ha−1 at topdressing (DG-N85 + Nm60)); (viii) application of digestate equivalent to 170 kg N ha−1 plus mineral nitrogen split in two applications (35 kg N ha–1 at preplant and 25 kg N ha−1 at topdressing (DG-N170 + Nm60)); (ix) application of digestate equivalent to 170 N ha−1 plus mineral nitrogen at 25 kg ha−1 at topdressing (DG-N170 + Nm25). Digestate was always applied at preplant. The mineral N at a rate of N of 85 kg ha–1 is the recommended N fertilizer rate for both crops [32]. The treatments were the same for the two crops. Table 2. Treatments and fertilizations performed during field experiments with lettuce (2020) and kale (2020–2021). Treatments 1st Crop—Lettuce 2nd Crop—Kale Preplant 1st topdressing 2nd topdressing Preplant 1st topdressing 2nd topdressing February 2020 March 2020 April 2020 October 2020 December 2020 February 2021 kg N ha−1 Control - - - - - - Ni85 Ni35 (CAN) Ni25 (CAN) Ni25 (CN) Ni35 (AS) Ni25 (AS) Ni25 (AS) Figure 1. Climate conditions during the experiments. 2.1. Experimental Design An experiment with a fully randomized design and four replications was performed. Each replication corresponded to a plot of 1.08 m 2 (1.20 × 0.9 m). The experiment was the same as reported by [ 31 ], and it involved nine treatments (Table 2): (i) no fertilization (control); (ii) mineral N fertilization at a rate of 85 kg ha −1 (Nm85) split in three applications (35 kg N ha −1 at preplant and two topdressings of 25 kg N ha −1 ); (iii) application of digestate (DG) equivalent to 85 kg N ha −1 (DG-N85); (iv) application of digestate equivalent to 170 kg N ha−1(DG-N170); (v) application of digestate equivalent to 255 kg N ha−1 (DG-N255); (vi) application of digestate equivalent to 340 kg N ha −1 (DG-N340); (vii) application of digestate equivalent to 85 kg N ha −1 plus mineral nitrogen split in two applications (35 kg N ha−1 at preplant and 25 kg N ha −1 at topdressing (DG-N85 + Nm60)); (viii) application of digestate equivalent to 170 kg N ha − 1 plus mineral nitrogen split in two applications (35 kg N ha–1 at preplant and 25 kg N ha −1 at topdressing (DG-N170 + Nm60)); (ix) application of digestate equivalent to 170 N ha −1 plus mineral nitrogen at 25 kg ha −1 at topdressing (DG-N170 + Nm25). Digestate was always applied at preplant. The mineral N at a rate of N of 85 kg ha –1 is the recommended N fertilizer rate for both crops [32]. The treatments were the same for the two crops. Both crops, lettuce and kale, were grown during two consecutive growing seasons (2020 and 2021). The digestate was manually incorporated into the soil 11 days before planting each crop. Then, plots were watered to maintain soil humidity at 80% field capacity, which is an adequate level for the correct growth of both crops [ 33 ]. Subsequently, two days before planting, preplant mineral N fertilizer was manually applied and incorporated into the soil. The lettuce had a crop cycle of 62 days (planted on 6 March 2020 and harvested on 7 May 2020). Topdressing N fertilization with mineral fertilizer was performed for the two crops at 22 and 49 days after planting or at the first date if only one topdressing was applied. The kale crop had a crop cycle of 152 days (planted on 15 October 2020 and harvested on 16 March 2021). Ammonium sulphate was used at preplant to reduce the risk of N leaching. The different mineral fertilizers and concentrations used in the experiment are described in Table 2. The soil for the experiment had a high level of available P (Olsen P = 149 mg kg −1 ) Agronomy 2023,13, 1308 4 of 16 and potassium (K = 530 mg kg −1 ). Therefore, there was not an expectable response to the application of P and K fertilizers, and only N fertilization was performed. Table 2. Treatments and fertilizations performed during field experiments with lettuce (2020) and kale (2020–2021). Treatments 1st Crop—Lettuce 2nd Crop—Kale Preplant 1st topdressing 2nd topdressing Preplant 1st topdressing 2nd topdressing February 2020 March 2020 April 2020 October 2020 December 2020 February 2021 kg N ha−1 Control - - - - - - Ni85 Ni35 (CAN) Ni25 (CAN) Ni25 (CN) Ni35 (AS) Ni25 (AS) Ni25 (AS) DG-N85 DG-85 - - DG-85 - - DG-N170 DG-170 - - DG-170 - - DG-N255 DG-255 - - DG-255 - - DG-N340 DG-340 - - DG-340 - - DG-N85 + Ni60 DG-85 + Ni35 (CAN) Ni25 (CAN) - DG-85 + Ni35(AS) Ni25 (AS) - DG-N170 + Ni60 DG-170 + Ni35 (CAN) Ni25 (CAN) - DG-170 + Ni35(AS) Ni25 (AS) - DG-N170 + Ni25 DG-170 Ni25 (CAN) - DG-170 Ni25 (AS) - Ni, mineral N, DG, digestate; CAN, calcium ammonium nitrate (27% Ni); CN, calcium nitrate (15.5% Ni); AS, ammonium sulphate (20.5% Ni). Source: Horta, C. and Carneiro, J.P. Use of Digestate as Organic Amendment and Source of Nitrogen to Vegetable Crops. Appl. Sci. 2022 , 12, 248. https://doi.org/10.3390/app12010248, Ref. [ 31 ]. 2.2. Digestate The digestate was produced as a by-product of the anaerobic digestion of a mixture of pig slurry and cereal straw. This digestion was performed during 30 days of retention time in mesophilic reactors with a capacity of 3000 m 3 . The digestate slurry was pressed to separate the solid and liquid phases. Only the solid fraction of the digestate was used in the present work. The chemical composition and biological properties of the solid digestate are shown in Table 3. The methods used in the characterization of digestates can be referenced in [ 31 ]. The total microbial biomass (TMB) of the digestates used in this experiment, estimated from the lipid acid profile, had a value of 552.67 nmol g −1 and was composed mainly of bacteria (135.49 nmol g −1 ). The ratio of bacteria:fungi was 17.11, and the ratio between Gram+ and Gram−bacteria was 1.95. Table 3. Chemical properties of the digestate (DG). DG Lettuce DG Kale DM (g kg−1)282 248 OM (g kg−1)637 761 pH (H2O) 7.7 8.3 EC (dS m−1)1.46 1.25 N-total (g kg−1)29.7 29.7 N-org (g kg−1)19.3 24.1 C/N 12 15 PT (g kg−1)4.8 7.1 Pi (g kg−1)4.5 6.8 Po (g kg−1)0.3 0.3 K (g kg−1)17 20.3 DM, dry matter; OM, organic matter; EC, electrical conductivity; N-org, organic nitrogen; PT, total phosphorus; Pi, inorganic phosphorus; Po, organic phosphorus. Agronomy 2023,13, 1308 5 of 16 2.3. Soil Analysis At the end of the experiments in May 2020 and March 2021, soil samples were taken for each repetition (0–20 cm depth) at 10 different locations and mixed to obtain a representative and unique sample. Each soil sample was divided into two fractions, one of which was immediately sieved to <2 mm and kept at 4 ◦ C for biological and biochemical analyses, while the other fraction was air-dried for chemical analyses. The pH was measured at a 1:2.5 soil:solution ratio, and the EC was measured with a conductivity meter (WTW, Weilheim, Germany) at a soil:water suspension ratio of 1:2. According to the procedure described by Walkley and Black, the organic matter was analyzed using a potentiometric titration method [ 34 ]. P was determined according to [ 35 ], and P in the solution was measured according to [ 36 ]. Total P (PT) was determined after ignition at 550 ◦ C for 3 h and extracted with H 2 SO 4 0.5 M, and inorganic P (Pi) was extracted with H 2 SO 4 0.5 M and then quantified by molecular absorption spectrophotometry. Organic P (Po) was calculated as the difference between PT and Pi. All the P in the extracts was centrifugated at 3000 rpm, and P in the supernatant was quantified using the method of Murphy and Riley [ 36 ]. The content of Nt was determined after the wet digestion process with sulfuric acid (H 2 SO 4 ) using the Kjeldahl method. Nitrate and ammonium were determined by ISO 14256-1 with 1 mol L −1 potassium chloride solution. For nitrate, all samples were below the detection limit according to this method. The methods used in the chemical analyses of the soil can be referenced in [ 31 ]. Enzymatic activities were determined after harvesting at the end of the second crop. β -glucosidase (Bglu) activity was determined according to the methods described in [ 37 ]. The activity of acid and alkaline phosphatase (AcPh and AlPh) was determined by measuring the amount of p-nitrophenol (PNP) released from the addition of 5 mM p-nitrophenylphosphate at pH 6.5 for AcPh and at pH 11 for AlPh, according to [38]. Total microbial biomass (TMB) was determined according to ester-linked fatty acids (ELFAs) after extraction in mild alkaline methanol, which is known to extract ester-linked fatty acids but not free fatty acids. First, 15 mL of 0.2 M KOH in methanol was added to 3 g of soil and incubated at 37 ◦ C for 1 h. The tubes were vortexed every 10 min during incubation. Then, the pH of the suspension was neutralized by adding 3 mL of 1 M acetic acid. ELFAs were separated into an organic phase by adding 10 mL of hexane, followed by centrifugation at 480 g for 10 min. After that, the hexane layer was transferred to a clean glass test tube, and the hexane evaporated with a rotavapor (Eppendorf AG concentrador plus, Hamburg, Germany) at 30 ◦ C for 20 min. Finally, ELFAs were dissolved in 0.5 mL of 1:1 hexane:methyl tert-butyl ether and transferred to a gas chromatograph (6890 N Network GC System, Agilent Technologies Wilmington, EEUU) vial for analysis. Four near infrared reflectance (NIR) spectra (one for each soil sample of each repetition) (MPA Bruker, Hetlingen, Germany) were obtained using reflectance light in a macrosample rotating integrated sphere. Samples were measured with a spectral resolution of 8 cm –1 , and the final spectra were obtained as the average of 64 scans from 4000 to 12,000 cm–1. 2.4. Data Analysis A heat map was carried out with the analytical data (chemical, enzymatic, and microbiological), aiming to have an overall evaluation of the influence of the different soil treatments. In this work, the classification tree was used to understand which variables were more representative to differentiate soil treatments. At each classification tree step, the most informative parameters were selected as the source of the (sub)tree, and the current training set was split into subsets according to the values of the selected attribute. The selected parameter was considered a good discriminator if the branches separated all the measurements observed for each sample group. To identify the effects of the treatments, ANOVA was performed. Normality was checked using the Kolmogorov–Smirnov test and homoscedasticity of variance with the Levene test [ 39 ]. The data were transformed (power or logarithmically) when one or both tests were not passed. Means were compared using Tukey’s test at p< 0.05. Agronomy 2023,13, 1308 6 of 16 To perform PCA with the spectral data collected with NIR, different mathematical preprocesses were used, namely the Savitzky–Golay first and second derivative, standard normal variate (SNV) transformation, multiplicative scatter correction (MSC), and different combinations of these treatments. Heat map and classification tree analyses were performed using Statistics ® version 7.0.0, the ANOVA with the statistical package Statgraphics Centurion XVI [ 40 ], and the spectral data analyses with the Unscrambler ® X software, version 10.5.46461.632 (CAMO Software AS, Oslo, Norway). 3. Results 3.1. Effect of Digestates on Soil Chemical Properties The ratio of organic N to total N (No:NT) was 0.65 for the digestates applied to the lettuce crop and 0.81 for the digestates applied to the kale crop, which means that most of the N was in organic forms. On the contrary, most of the P was in inorganic forms, with a ratio between inorganic P and total P (Pi:PT) of 0.94 for the digestates used in both seasons. (Table 3). The applied digestate provided P rates ranging from 13.7 to 54.9 kg P ha –1 . In addition, K, Ca, and Mg were supplied by the digestates in significant amounts (Table 3). The total N in the soil increased significantly with the application of digestates in both crops. After both crops, the highest soil total N was achieved with the treatments providing the highest total N rate (DG-N340 and DG-N255, 3.5 and 3.97 g kg −1 , respectively). The average NH 4+ -N was 42% higher after the lettuce crop (0.88 g kg −1 ) than the kale crop (0.50 g kg−1) (Tables 4and 5). This is explained in part by the high initial NH 4+ -N concentration or release from organic N mineralization in the initial soil, as can be seen from the control plots after the lettuce crop (Table 4). The high content of organic matter produces a supply of N to the soil due to its mineralization of approximately 85 kg N ha –1 , estimated with the mineral N content of the soil, mineral N at the end of the crop, and total N uptake by the plants in the control plots. Table 4. Soil properties after lettuce crop. Treatment Nt Nam Olsen P Po Pi SOM pH EC g kg−1mg kg−1% dS m−1 DG-N85 2.46 cd a) 0.88 b 100.73 cd 253 768 ab 4.6 6.15 ab 0.11 c DG-N170 2.63 bc 0.86 b 111.83 bc 275 827 a 7.87 6.28 a 0.12 bc DG-N255 2.94 ab 0.89 b 132.1 a 272 852 a 6.70 6.31 a 0.15 ab DG-N340 3.08 a 1.06 ab 123.76 ab 267 835 a 6.96 6.31 a 0.15 ab DG-N85 + Ni60 2.82 ab 0.81 b 96.4 cd 293 734 ab 6.42 5.96 c 0.11 c DG-N170 + Ni60 2.88 ab 1.21 a 103.15 cd 336 794 ab 7.47 6.08 bc 0.12 bc DG-N170 + Ni25 2.8 ab 0.90 b 102.14 cd 254 745 ab 5.48 6.24 ab 0.12 bc Ni85 2.36 cd 1.28 a 94.17 cd 302 666 b 4.31 5.72 d 0.19 a Control 2.27 d 1.04 ab 88.88 d 300 655 b 5.11 6.08 bc 0.11 c ANOVA * ** *** NS *** NS *** *** Nt, total nitrogen; Nam, ammonium nitrogen; Po, organic phosphorus; Pi, inorganic phosphorus; SOM, soil organic matter; EC, electrical conductivity. *, **, and *** Significant at p< 0.05, p< 0.01, and p< 0.001, respectively, and NS-not significant. a) Different letters in the same column indicate that the means (n= 4) were significantly different according to Tukey’s test at a probability level of 0.05 for the treatments. Organic P did not show significant differences between treatments after lettuce, with values in the range of 253–336 mg kg −1 . On the contrary, after the kale crop, the organic P content in the soil amended with digestates at all rates was higher (on average 38%) than that with mineral fertilizer and that in the nonfertilized control (on average 48%, Table 5). The inorganic P measured in the soil after the lettuce crop increased with digestate application compared to Nm fertilizer treatment and the nonfertilized control (Table 4). Agronomy 2023,13, 1308 7 of 16 Table 5. Soil properties after kale crop. Treatment Nt Nam Olsen P Pi Po SOM pH EC g kg−1mg kg−1% dS m−1 DG-N85 2.93 ab a) 0.64 ab 77.27 cde 596 cd 269 abc 7.61 abc 6.38 c 0.08 b DG-N170 2.85 ab 0.66 ab 83.51 bcde 692 bc 443 a 7.87 abc 6.53 b 0.09 b DG-N255 3.97 a 0.78 a 109.91 a 806 a 376 ab 8.22 a 6.66 ab 0.1 ab DG-N340 3.50 ab 0.41 ab 107.98 a 759 ab 372 abc 8.26 a 6.70 a 0.12 a DG-N85 + Ni60 3.08 ab 0.67 a 85.46 bcd 685 bc 451 a 8.01 ab 6.08 d 0.07 b DG-N170 + Ni60 3.67 ab 0.60 ab 96.10 ab 729 ab 352 abc 7.85 abc 6.11 d 0.09 b DG-N170 + Ni25 2.83 ab 0.39 ab 91.06 bc 680 bc 237 bc 7.29 abc 6.27 c 0.08 b Ni85 2.36 b 0.31 ab 73.33 de 539 d 219 bc 6.83 c 5.91 e 0.07 b Control 2.59 ab 0.13 b 66.98 e 545 d 185 c 7.10 bc 6.27 c 0.08 b ANOVA * ** *** *** *** ** *** *** Nt, total nitrogen; Nam, ammonium nitrogen; Po, organic phosphorus; Pi, inorganic phosphorus; SOM, soil organic matter; EC, electrical conductivity. *, **, and *** Significant at p< 0.05, p< 0.01, and p< 0.001, respectively. a) Different letters in the same column indicate that the means (n= 4) were significantly different according to Tukey’s test at a probability level of 0.05 for the treatments. Digestate application in the lettuce crop at rates of 170 kg N ha −1 or higher showed the highest inorganic P values (827–852 mg kg −1 ) (Table 4); on average, these three DG treatments showed an inorganic P concentration 28% higher than the treatment in the control without fertilizer and 26% higher than mineral N fertilizer treatment. The inorganic P content after the lettuce crop showed a mean value (764 mg kg −1 ) higher than that after the second crop with kale (670 mg kg −1 ) (Tables 4and 5). After the second year with kale, DG-N255 showed the highest content of inorganic P in the soil, with a mean value of 806 mg kg−1, which was 48% higher than the nonfertilized control and 50% higher than with mineral N fertilizer (Table 5). The P available in the soil measured as Olsen P showed a similar trend to inorganic P (Tables 4and 5). Olsen P after the first crop was significantly increased with the application of the two highest digestate rates relative to mineral or not fertilized treatments, with the highest value obtained with DG-N255 (132.10 mg kg −1 ; Table 4). A similar trend was observed after the second crop, with DG-N340 and DG-N255 being the treatments with the highest Olsen P (108.94 mg kg−1) (Table 5). Digestates applied alone significantly increased pH values relative to the nonfertilized control or treatments involving the application of mineral N. The highest digestate rate increased the pH to 6.3 and 6.75 after lettuce and kale, respectively. Overall, pH increased with increased digestate rate, while the lowest pH values were obtained with mineral N fertilizer (Nm 85), at 5.72 and 5.91 after lettuce and kale, respectively (Tables 4and 5). As expected, the soil organic matter (SOM) content in soil increased with increased digestate rates, but the differences were only significant after the kale crop. After the lettuce crop, SOM varied in the range of 4.31–7.87% SOM, which were lower values than after the kale crop (6.95–8.30%) (Tables 4and 5). At the end of the two crop cycles, DG-N255 and DG-N340 increased SOM by 17% relative to the nonfertilized control (Table 5). 3.2. Effect of Digestates on Soil Biochemical and Biological Properties The digestates at the highest rate decreased acid phosphatase and β -glucosidase activities compared to the nonfertilized control, and the three highest digestate rates decreased alkaline phosphatase relative to the mineral N fertilizer treatment (Table 6). Alkaline phosphatase activity was negatively correlated with inorganic P in soil (r = −0.5; p< 0.01) and positively correlated with total microbial biomass (TMB; r = 0.55; p< 0.001). Overall, digestate application decreased TMB as well as fungi and bacteria populations relative to the mineral N fertilizer treatment and the nonfertilized control (Table 6). Agronomy 2023,13, 1308 8 of 16 Table 6. Total microbial biomass and microbial communities in soil after two consecutive crops. Treatment AcPh AlPh B-glu TMB Bacteria Fungi BacG+ BacG- Mycorrhizae mg pnp kg−1h−1nmol g−1 DG-N85 495 ab a) 179 ab 164 ab 262 ab 53.7 ab 19.0 ab 24.0 abc 20.9 ab 11.2 ab DG-N170 455 bc 144 bc 140 b 225 c 48.1 b 17.2 ab 21.6 bc 20.8 ab 7.9 ab DG-N255 480 ab 153 bc 159 ab 224 c 46.8 b 14.2 b 20.1 c 21.3 ab 6.6 ab DG-N340 415 c 125 c 124 b 230 bc 48.4 b 15.0 b 21.7 bc 18.0 b 8.4 b DG-N85 + Ni60 461 abc 144 bc 141 b 218 c 46.3 b 15.8 b 21.0 bc 19.9 b 8.2 ab DG-N170 + Ni60 520 a 170 ab 171 ab 231 bc 50.1 ab 15.1 ab 21.4 bc 18.8 ab 8.1 ab DG-N170 + Ni25 487 ab 130 c 170 ab 241 bc 48.2 b 17.8 ab 20.9 bc 19.7 ab 8.8 ab Ni85 462 abc 201 a 204 a 281 a 57.6 a 20.9 a 26.7 a 21.2 ab 10.4 ab Control 484 ab 160 bc 197 a 295 a 57.0 a 20.6 a 24.8 ab 25.0 a 11.5 a ANOVA ** *** *** *** *** ** *** * AcPh, acid phosphatase; AlPh, alkaline phosphatase; TMB, total microbial biomass; BacG+, bacteria Gram+; BacG − , bacteria Gram − . *, **, and *** Significant at p< 0.05, p< 0.01, and p< 0.001, respectively. a) Different letters in the same column indicate that the means (n= 4) were significantly different according to Tukey’s test at a probability level of 0.05 for the treatments. However, the highest digestate rate did not lead to a lower mycorrhizae population compared to the nonfertilized control (Table 6). 3.3. Clustering the Effect of Fertilizer Treatments on Soil The heat map (Figure 2) showed that the treatments can be organized into two big clusters (Cluster 1 group 2) with different behaviors regarding their correlations with the chemical or enzymatic and microbiological soil properties. Concerning the treatments, the first group was composed of the control, Ni85, DG-N85, and DG-N170 + Nm25, which showed overall positive correlations between enzymatic and microbiological properties and negative correlations with soil chemical properties. The second group was formed by all the other treatments (DG-N170, DG-N170 + Nm60, DG-N85 + Nm60, DG-N255, and DG-N340) and was shown to have correlations with opposite trends. Agronomy 2023, 13, x FOR PEER REVIEW 9 of 17 Figure 2. Heatmap of the correlations and tendencies between chemical, enzymatic, and microbiological data and soil treatments. Regarding the classification tree (Figure 3), acid phosphatase discriminates DG-N340 from all the other treatments. Then, the level of soil total P discriminates two groups: the first group was composed of DG-N85, DG-N170 + Nm25, Ni85, and the control; and the second group was composed of DG-N170, DG-N255, DG-N85 + Nm60, and DG-N170 + Nm60. Next, node 4 with Gram+ bacteria discriminate DG-N170 + Nm25, and Olsen P discriminates DG-N85, Nm85, and the control. Node 5, using the soil Olsen P level, discriminates treatments DG-N170, DG-N85 + Nm60, DG-N170 + Nm60, and DG-N255. Node 7 discriminates treatments though the soil total P into DGN85 and another group consisting of Nm85 and the control. Node 10 discriminates these two last treatments through AlPh. Node 8 also used alkaline phosphatase activity to discriminate between DG-N170 and DG-N85 + Nm60 from DG-N170 + Nm60. Note that the treatments DG- N170 and DG-N85 + Nm60 cannot be separated by any soil property, suggesting that they have a similar effect on soil. Figure 2. Heatmap of the correlations and tendencies between chemical, enzymatic, and microbiological data and soil treatments. Agronomy 2023,13, 1308 9 of 16 Regarding the classification tree (Figure 3), acid phosphatase discriminates DG-N340 from all the other treatments. Then, the level of soil total P discriminates two groups: the first group was composed of DG-N85, DG-N170 + Nm25, Ni85, and the control; and the second group was composed of DG-N170, DG-N255, DG-N85 + Nm60, and DG-N170 + Nm60. Next, node 4 with Gram+ bacteria discriminate DG-N170 + Nm25, and Olsen P discriminates DG-N85, Nm85, and the control. Node 5, using the soil Olsen P level, discriminates treatments DG-N170, DG-N85 + Nm60, DG-N170 + Nm60, and DG-N255. Node 7 discriminates treatments though the soil total P into DGN85 and another group consisting of Nm85 and the control. Node 10 discriminates these two last treatments through AlPh. Node 8 also used alkaline phosphatase activity to discriminate between DG-N170 and DG-N85 + Nm60 from DG-N170 + Nm60. Note that the treatments DG-N170 and DG-N85 + Nm60 cannot be separated by any soil property, suggesting that they have a similar effect on soil. Agronomy 2023, 13, x FOR PEER REVIEW 10 of 17 Figure 3. Classification tree results for the chemical, enzymatic, and microbiological data with soil properties using Gini methodology. 3.4. Effect of Digestate on Soil Spectral Traits The NIR spectra of all the samples that were analyzed had similar profiles, with the main typical and important absorbance bands of soil at 4512, 5200, and 7054 cm−1 [41]. The spectral peaks around 5200 cm−1 and 7054 cm−1 correspond to the water absorption bands and can be assigned to the first and second overtones of -OH stretching. These two peaks were strongly affected by soil moisture; however, prior analysis of the soil it was dried until constant moisture. These bands can also be associated with organic matter, clay minerals, and the carbonyl group (C=O) in organic matter [42,43]. The peak around 4530 cm−1 could be associated with organic functional groups of organic matter in the soil (C-H bonds of aliphatic compounds), OH-, and Al-OH bonds of clay minerals [44,45]. The same NIR region was used by [46] to assess soil quality and classify sites according to their global degradation status and by [47] to predict total C, organic C, total N, and moisture content of soils from different geographic regions. The correlation between soil samples obtained with PCA was quite similar to the results obtained with the heat map, but there were some differences, because with NIR we have information about all soil constituents and not only on the parameters measured in this work. Nevertheless, it is possible to plot that the most relevant regions for distinguishing the samples analyzed were from 4000 cm−1 to 5450 cm−1 and from 6970 cm−1 to 7347 cm−1 (Figure 4). PCA analysis showed that component 1 explained 74% of the total variability. This component clearly distinguishes the control group and N85 (the treatments without any fertilization or without DG) from the other groups with higher levels of DG application (DG-N255 and DG-N340). The other samples showed intermediate behavior. Component 2, which explains 30% of the total variation, distinguishes the control treatment from N85. This component is also responsible for the discrimination of DGN85 from DG-N170 + Nm60 (Figure 4). DG-N85 DG-N170 DG-N255 DG-N340 DG-N85+Nm60 DG-N170+Nm60 DG-N170+Nm25 Nm85 control 1 2 3 4 5 6789 10 11 12 13 14 15 AcPh ≤ 429.25 Pt ≤ 970.28 BacG+ ≤ 22.275 Olsen-P ≤ 101.75 Pt ≤ 816.97 AlPH ≤ 151.34 AlPH ≤ 175.38 432 16 16 412 12 4 8 4 8 4 4 4 DG-N340 DG-N170+Nm25 DG-N255 DG-N85 DG-N170 DG-N85+Nm60 DG-N170+Nm60 control Nm85 Figure 3. Classification tree results for the chemical, enzymatic, and microbiological data with soil properties using Gini methodology. 3.4. Effect of Digestate on Soil Spectral Traits The NIR spectra of all the samples that were analyzed had similar profiles, with the main typical and important absorbance bands of soil at 4512, 5200, and 7054 cm −1 [ 41 ]. The spectral peaks around 5200 cm −1 and 7054 cm −1 correspond to the water absorption bands and can be assigned to the first and second overtones of -OH stretching. These two peaks were strongly affected by soil moisture; however, prior analysis of the soil it was dried until constant moisture. These bands can also be associated with organic matter, clay minerals, and the carbonyl group (C=O) in organic matter [42,43]. The peak around 4530 cm −1 could be associated with organic functional groups of organic matter in the soil (C-H bonds of aliphatic compounds), OH-, and Al-OH bonds of clay minerals [ 44 , 45 ]. The same NIR region was used by [ 46 ] to assess soil quality and classify sites according to their global degradation status and by [ 47 ] to predict total C, organic C, total N, and moisture content of soils from different geographic regions. Agronomy 2023,13, 1308 16 of 16 62. Thomsen, I.K.; Olesen, J.E.; Møller, H.B.; Sørensen, P.; Christensen, B.T. Carbon dynamics and retention in soil after anaerobic digestion of dairy cattle feed and faeces. Soil Biol. Biochem. 2013,58, 82–87. [CrossRef] 63. Häfner, F.; Hartung, J.; Möller, K. Digestate Composition Affecting N Fertiliser Value and C Mineralisation. Waste Biomass Valor. 2022,13, 3445–3462. [CrossRef] 64. 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