Effect of biochar and compost on soil properties and organic matter in aggregate size fractions under field conditions

Agriculture, Ecosystems and Environment 295 (2020) 106882

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Effect of biochar and compost on soil properties and organic matter in aggregate size fractions under field conditions

T

Jennifer Coopera,*, Isabel Greenbergb, Bernard Ludwigb, Laura Hippichb, Daniel Fischerc, Bruno Glaserc, Michael Kaisera a

University of Lincoln-Nebraska, Department of Agronomy and Horticulture, 202 Keim Hall, Lincoln, NE, 68583-0915, USA University of Kassel, Department of Environmental Chemistry, Nordbahnhofstrasse 1a, 37213, Witzenhausen, Germany c Martin Luther University Halle-Wittenberg, Institute of Agronomy and Nutritional Sciences, Soil Biogeochemistry, von-Seckendorff-Platz 3, D-06120, Halle, Germany b

A R T I C LE I N FO

A B S T R A C T

Keywords: Soil carbon storage Soil aggregation Biochar Compost Co-Composting Field experiment

Aggregation affects soil properties crucial for sustainable soil management and productivity. However, longerterm studies (> five years) of treatments to enhance soil aggregation, such as addition of biochar with labile carbon to derive microbial binding agents, are limited, especially in temperate climates. To fill this gap, we established a field experiment with control, compost only, biochar only, and a mixed compost-biochar application (co-composted and only mixed) at low and high application rates (9–70 t ha−1) in southern Germany. After six years of agricultural use, surface (0−10 cm) and subsurface (10−30 cm) soil samples were analyzed for pH, microbial biomass carbon (Cmic), water holding capacity (WHC), and cation exchange capacity (CEC). Particulate organic matter (POM) and aggregate fractions were analyzed for organic carbon (OC) content and characterized using diffuse reflectance infrared Fourier transform spectroscopy. Biochar significantly increased OC and pH, while compost significantly increased OC, pH, Cmic, and CEC six years after application. Biochar also significantly increased OC storage in POM (10–32 times higher) and all aggregate fractions by 56–62 %, 32–47 %, and 29–32 % for the 2 - 0.25 mm, 0.25 - 0.053 mm, and < 0.053 mm fraction, respectively, while compost increased OC storage only in the 10−30 cm soil depth in the soil fractions > 0.053 mm. The proportion of reactive C]O groups significantly increased in POM due to biochar and compost application, while only biochar affected the < 0.053 mm fraction. Our results suggest that six years after application, high rates of both biochar and compost are beneficial for soil properties affecting the sustainability of soil agro-ecosystems such as pH and CEC. For long lasting increase in soil C sequestration, our results indicate that only the application of biochar can be considered as a significant measure.

1. Introduction Biochar application to soil has gained increasing attention for more than a decade (Arthur et al., 2015) following the discovery of highly fertile Terra Preta (Verheijen et al., 2010). Terra Preta is anthropogenic Amazonian dark soil cultivated by indigenous tribes hundreds of years ago. Part of their cultivation practice was to add charred biomass (i.e. biochar) to soil, in addition to excrements and composted food leftovers (Glaser and Birk, 2012). In the evolved Terra Preta, researchers have found unusually nutritious soils with much higher carbon (C) contents as compared with adjacent Ferralsols that received no additions (Glaser et al., 2001). It has been shown that charred residues are key for Terra Preta genesis as black C contents are enriched in Terra Preta by a factor

of 70, while organic matter (OM) is enriched only threefold (Glaser et al., 2001; Glaser, 2007; Glaser and Birk, 2012). Biochar is produced by pyrolysis, a process during which biomass is heated under oxygen-limited conditions. During pyrolysis, temperatures up to 1000 °C are reached, water is evaporated, and biomass is converted into char. The feedstock used for this process is C-rich biomass, such as wood, manure or crop residues (Verheijen et al., 2010). Pyrolysis temperature, feedstock and process time are the main factors controlling physical and chemical properties of the produced biochar (e.g. Schimmelpfennig and Glaser, 2012; Weber and Quicker, 2018). These physical and chemical properties include high C-content (> 70 %), fine or coarse pore structure, high surface area (259–532 m2 g−1), high initial pH (commonly > 7), and high CEC (10–70 cmolc kg−1)

⁎ Corresponding author at: University of Nebraska-Lincoln, Department of Agronomy & Horticulture, 1875 N 38thSt, 279 Plant Sciences Hall, PO Box 830915, Lincoln, NE, 68583-0915, USA. E-mail address: [email protected] (J. Cooper).

https://doi.org/10.1016/j.agee.2020.106882 Received 16 October 2019; Received in revised form 19 February 2020; Accepted 21 February 2020 0167-8809/ © 2020 Elsevier B.V. All rights reserved.

Agriculture, Ecosystems and Environment 295 (2020) 106882

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effects of biochar in temperate soils, we sampled a field experiment in SW Germany where biochar in combination with compost was applied. The treatments included the addition of compost only, biochar only, and a compost-biochar mixture at two application rates, both with and without co-composting. Soil samples were taken from 0−10 cm (surface) and 10−30 cm (subsurface), and separated into four aggregate size fractions. The aggregate size fractions were analyzed for OC and characterized by infrared spectroscopy. Due to its higher reactivity, we expected increases in CEC, WHC, pH, and microbial biomass C (Cmic) to result primarily from the compost additions, while OC storage was expected to be more influenced by biochar additions. Furthermore, compared to biochar or compost only additions, we expected soils receiving compost-biochar mixtures to result in the greatest increase in aggregate formation and OC storage because compost is reactive, and might act as an effective aggregate binding agent for biochar-derived OC. The co-composting is assumed to further increase this positive effect because biochar is expected to become increasingly reactive due to potential biogeochemical reactions and organic coating (i.e. aging) of its surface.

(Chan and Xu, 2009; Mukherjee et al., 2011; Yu et al., 2006). As a result of pyrolysis, part of the biomass is converted into a highly aromatic form, depending on the feedstock and process parameters. The altered physical and chemical characteristics contribute to the low degradation rate and thus relatively high residence times of biochar in soil (Verheijen et al., 2010). The decelerated degradation of biochar-derived organic C (OC) in soil can significantly increase the long-term C sequestration in agro-ecosystems and mitigate increasing atmospheric CO2 (Kuzyakov et al., 2014; Lehmann, 2007; Smith, 2016). Besides its relevance for soil OC storage, positive effects of biochar on productivity-relevant soil chemical properties such as cation exchange capacity (CEC) have been detected in both incubation (e.g. Van Zwieten et al., 2010) and field experiments (e.g. Major et al., 2010). As surface oxidation and, therefore, CEC increase over time (Cheng et al., 2006 and 2008), biochar carries a potential for long-term enhancement of nutrient storage and supply. Increased soil nutrient availability and plant productivity due to the application of biochar is also attributed to its liming effect (Chan and Xu, 2009) and the accompanied rise in soil pH (Major et al., 2010; Rondon et al., 2007). Additionally, because of its pore structure, biochar further acts as a habitat for soil microorganisms (Verheijen et al., 2010) leading, in conjunction with increased nutrient availability and pH, to enhanced microbial activity (Lehmann et al., 2011) and the usage of biochar as a substrate (Pietikäinen et al., 2000). The effects of biochar on soil physical properties, such as the stability and formation of aggregates (e.g. Liu et al., 2012b; Ouyang et al., 2013), have been explored to a lesser extent. Soil aggregation in turn affects numerous soil functions such as water holding capacity (WHC), storage of OM and nutrients, and soil resilience to climatic and mechanical stresses and is, therefore, of great importance for crop development and yields. However, studies focusing on the effects of biochar on soil aggregation under agricultural field conditions are scarce (Haider et al., 2017), and most do not last more than few months (Liu et al., 2012b). Due to its reactive functional groups (OH, COOH), biochar is generally expected to engage in bonding interactions with binding agents and promote aggregation at different scales in soil (Kaiser et al., 2014), which might even increase during aging of biochar (Mukherjee et al., 2014). However, conflicting results on water-stable aggregate formation after biochar addition have been reported (Mukherjee and Lal, 2013). In pot experiments, both enhanced macro-aggregate formation (Ouyang et al., 2013; Sun and Lu, 2014) as well as no effects on soil structure were found (Grunwald et al., 2016). It has been suggested that aggregate formation, which is highly dependent on availability of OM, could be enhanced if biochar is incorporated in combination with a labile OM source, such as compost, in order to strengthen microbial communities and the derived binding agents (Mukherjee and Lal, 2013). This would also correspond with the Terra Preta model, where biochar was added together with easily degradable biomass such as excrement and compost (Glaser and Birk, 2012). A more labile OM source could be compost, i.e. organic material decomposed under aerobic conditions by microorganisms, which might induce positive synergistic effects in combination with biochar to create a nutrient-rich organic substrate with terra preta-like properties (Fischer and Glaser, 2012). Several studies have examined effects of adding biochar together with organic (Liu et al., 2012a) or mineral fertilizer (Jones et al., 2012), as well as co-composted biochar (Agegnehu et al., 2015; Glaser et al., 2015), but mainly focused on pH, CEC and WHC, and crop yield. Therefore, a knowledge gap exists regarding soil aggregate formation and stability following addition of both biochar and compost under longer-term field conditions (i.e. > 5 years) (Tammeorg et al., 2017). Furthermore, most field studies were not conducted under temperate climatic conditions, which is a pre-requisite for the implementation of large-scale addition of biochar to agricultural ecosystems in Europe. To fill the identified knowledge gaps on long-term (i.e. > 5 years)

2. Material and methods 2.1. Site description and field experimental design The field experiment is located in Donndorf / Eckersdorf (near Bayreuth, Germany) in northeastern Bavaria and was established in 2010 to analyze the effects of different soil amendments consisting of biochar and/or compost on soil properties and crop yield (Fischer, 2014). The climate is temperate, with temperature range of 5–13 °C, and a mean annual precipitation of 507 mm. The experimental site covers a total area of 3600 m² (30 m x 120 m) and consists of fifty individual plots of 72 m² (6 m x 12 m) each. The parent material is sand stone and the soil type is a Cambisol. With an average of 62 % sand, 12 % silt, and 26 % clay for bulk soil sampled at the 0−30 cm depth, the soil was classified as a sandy loam. The plots were arranged according to a Latin rectangle in a row-column design so that each treatment was present in each row and each pair of columns in a grid across the field. Each of the nine treatments was replicated five times and received a one-time amendment application in 2010. Treatments included a control without any application of compost or biochar, application of 20 or 70 t ha−1 of compost (C20 and C70), application of 9 or 31.5 t ha−1 of biochar (B9 and B31.5), application of compost-biochar mixtures where biochar and compost were mixed on site directly before being applied (C20B9 and C70B31.5), and application of compost-biochar mixtures where biochar and compost were mixed and co-composted for two months before application (C20B9 comp and C70B31.5 comp). This resulted in total C application rates of 7.6 t C ha−1 for B9, 26.6 t C ha−1 for B31.5, 3.7 t C ha−1 for C20, 13.0 t C ha−1 for C70, 11.3 t C ha−1 for C20B9, and 39.6 t C ha−1 for C70B31.5. The basic properties of the treatments are presented in Table 1. The commercially available biochar was derived from beech and pine wood chips (CarbonTerra, Wallerstein, Germany). Low-temperature pyrolysis (< 550 °C) lasted 36 h with short-duration high-temperature pyrolysis (700–800 °C) for 1–2 hours. After pyrolysis, glowing coals were quenched with water and stored in bags after cooling, and sieved to < 5 mm prior to application. Compost was derived from composted green litter. The soil amendments were distributed manually and homogenously on each plot and plowed to a shallow depth of 10 cm using a rotary harrow. Shallow tillage of the topsoil ( ± 10 cm) with a file cultivator/grubber was performed annually; however, the soil was not turned during tillage. During the field experiment, the crops grown on all treatments were millet (2010), ryegrass and corn (2011), triticale and mustard (2012), corn (2013), winter wheat and mustard (2014), corn (2015), and winter wheat (2016). Following harvest, the straw was removed from the soil. Organic fertilizers were applied with a liquid manure spreader (biogas corn digestate) or by broadcasting (cow 2

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Table 1 Basic properties of treatments used for application. Means are shown for total carbon (TC), total nitrogen (TN), C/N ratio, H/C and O/C atomic ratios, ash, pH, cation exchange capacity (CEC) and water holding capacity (WHC).

Compost Biochar Compost-biochar mixture

TC

TN

C/N

H/C

O/C

Ash

pH (CaCl2)

CEC

WHC

— [%] — 18.6 84.3 41.1

1.0 0.4 0.8

18.8 239.2 50.5

— [at %] — 1.41 0.11 0.52

0.62 0.03 0.19

[%] 66.1 8.8 46.5

6.99 8.77 7.60

[mmolc kg−1] 304.3 72.7 241.1

[%] 203.8 249.4 206.5

oxide, 10 mm diameter, frequency 30 Hz) for 2 min. Subsequently, samples were measured by dry combustion using a CN elemental analyzer (Elementar Vario El, HARAEUS, Hanau, Germany).

or horse slurry) to all treatments until 2016, when calcium ammonium nitrate fertilizer was applied using a twin disc spreader. 2.2. Soil sampling

2.4. Infrared spectroscopic analysis Soil samples were taken from the 0−10 cm and 10−30 cm depth in August 2016 after cereal harvest. To avoid effects from adjacent plots, samples were taken from the core area of each plot. For analysis of aggregate dynamics, each plot and depth was sampled three times using an auger. The samples were mixed into one composite sample and stored field-moist with care taken to avoid compression of the samples to preserve the original aggregate structure. Furthermore, samples were taken from each depth segment using a soil rod with attached sampling rings (100 cm³, height 4 cm). Biochar particles were distinguishable during sampling.

The OM composition of the dried and ball-milled POM and < 0.053 mm aggregate fractions for each sample were analyzed using Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy. These two fractions were chosen to capture the theoretically quickest (POM) and slowest (< 0.053 mm) fractions to incorporate organic carbon additions to the soil. The spectra of each sample was recorded with 200 scans in the wavenumber range of 400 to 4000 cm−1 (Tensor 27 and OPUS 7.2 software, BRUCKER, Ettlingen, Germany). Spectral information was parameterized by relating the added signal heights of two peaks in the “aliphatic” region of the spectra representing CH stretching (Band “A”, 2934 ± 28 and 2849 ± 14 cm−1) to the signal height in the ionizable “carbonyl” and “carboxyl” region representing C]O functional groups (Band “B”, 1610 ± 30 cm−1) (Kaiser et al., 2014; Ellerbrock and Gerke, 2013). The latter is indicative of the presence of groups contributing to the CEC (carboxyl groups) as well as the hydrophilicity of the OM. To facilitate a semi-quantitative comparison of treatments, B / A * OC was calculated as a measure of the relative amounts of C]O groups, whereby multiplication by OC scales the spectral information to the amount of OC in g per kg soil.

2.3. Laboratory analysis The pH value was measured using a 0.01 M CaCl2 solution (60 ml per 6 g soil sieved to < 2 mm, 30 min. shaking and 10 min. sedimentation). Microbial biomass C (Cmic) was determined by ChloroformFumigation-Extraction method using field moist samples (Joergensen, 1996). Soil texture was analyzed using the pipette method (DIN ISO 11277, 2002) following routine pre-treatment with hydroxide peroxide and hydrochloric acid and dispersion with sodium hexa-metaphosphate. The WHC was determined by saturation of undisturbed cores. Elements for the determination of CEC (effective) were measured by ion chromatography (Metrohm, Herisau, Switzerland) after extraction with a 0.1 M BaCl2 solution. The field moist soil samples for aggregate fractionation were carefully crushed and passed through a 10 mm sieve. Stones and organic residues > 10 mm were removed. To evaluate the aggregate-size class distribution, the samples were wet-sieved according to Jacobs et al. (2009). For this purpose, 100 g of < 10 mm dried (40 °C for 2 days) soil of each sample were evenly placed on a 2 mm sieve and carefully submerged in distilled water to minimize aggregate dispersion by slaking. After 10 min of soaking, the sieve was carefully moved vertically 50 times, immersing the sample completely with each repetition. After the first sieving, floating particulate organic matter (POM) was skimmed off and separately stored. The fraction > 2 mm remaining on the sieve surface was separated (fraction was stored and weighed) and the material which passed through the sieve was then transferred to a 0.250 mm sieve, where the procedure was repeated. The fraction smaller 0.250 mm was then transferred on a sieve with a mesh size of 0.053 mm and the sieving procedure was repeated. The wet-sieving procedure resulted in the separation of five fractions per sample: POM, 10 - 2 mm, 2 - 0.250 mm, 0.250 - 0.053 mm and < 0.053 mm. Each fraction was oven dried at 40 °C for 2 days. Aggregate yields were reported as mass percentages of the total bulk soil < 10 mm. For total carbon (Ct) analyses, bulk soil samples (10 g) and the aggregate size fractions of 2 - 0.250 mm (7 g) were ball-milled (MM400, RETSCH, Haan, Germany) using 10 balls (zirconium oxide, 10 mm diameter, frequency 30 Hz for 5 min). Due to small amounts of material recovered, the POM fraction and aggregate size fractions of 0.250 0.053 mm and < 0.053 mm were ball-milled using five balls (zirconium

2.5. Statistical analysis Statistics were carried out with the statistical software package R (Version 3.4.4; R Core Team, 2018). To ensure that parametric assumptions were met, the Shapiro-Wilk test for normality of residuals was conducted, residuals were visually inspected for homogeneity of variance, and the ratio of the maximum to the minimum standard deviation of the factor levels of each model factor was examined. Box-cox transformations were applied as needed in order to meet parametric assumptions (please see supplemental material for specific information). In the first part of a two-part statistical analysis, analysis of variance (ANOVA) was carried out to determine the effect of the factors compost and biochar on the soil response variables by considering the following 7 treatments: Control, C20, C70, B9, B31.5, C20B9 (no co-composting), and C70B31.5 (no co-composting). For this, we used two-way ANOVAs examining the effect of categorical factors compost (0, 20, 70 tons ha-1: no, low, high), biochar (0, 9, 31.5 tons ha-1: no, low, high), and their interaction, as well as column and row in the field due to the Latin rectangle design (Welham, 2015). ANOVAs were carried out separately for each soil depth. Stepwise model reductions were carried out, eliminating first a non-significant interaction, then non-significant main effects (Crawley, 2012), independent of the design factors row and column in the field. Since the experimental design is unbalanced, the order of the treatment factors in the model was varied, while stepwise model reductions were carried out to ensure correct ANOVA results (Mead et al., 2002). Tukey’s HSD test was applied to conduct pairwise comparisons in the case of a significant (p ≤ 0.05) treatment affect in the ANOVA. In a second, balanced two-way ANOVA, we evaluated the factors co3

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detected a significant interaction effect of the factors biochar and compost on WHC for the surface soil and a significantly higher WHC for the high-rate versus low-rate compost treatments in the subsurface soil between the low and high addition rates (Fig. 1, Table S2). The Cmic content was higher in surface soil (313–395 μg g−1) than in subsurface soil (214–285 μg g−1), with the highest Cmic content detected for the C70B31.5 treatment and the lowest for the B9 treatment at both soil depths (Fig. 1). Cmic was only significantly affected by the factor compost in the subsurface soil (control = low-rate < high-rate; Fig. 1, Table S2). The pH in both soil depths was slightly acidic, and ranged from 5.35 (surface control) to 5.84 (subsurface C70B31.5) (Fig. 1). The pH was significantly increased by biochar and compost in the surface soil (high-rate > control) but only by the factor compost in the subsurface soil (high-rate > low-rate = control; Fig. 1, Table S2). In both soil depths, the C70B31.5 treatment showed the highest mean CEC (134 and 135 mmolc) and the lowest CEC was found for C20B9 (92 mmolc) in the surface soil and for B9 (97 mmolc) in subsurface soil (Fig. 1). While biochar had no significant effect, we found a significant increase in CEC in response to compost (surface: control < low-rate < high-rate; subsurface: control = low-rate < high-rate; Fig. 1, Table S2). We found higher bulk soil OC contents in the surface soil (18.5–27.4 g OC kg−1 soil) as compared to the subsurface soil (14.0–24.0 g OC kg−1 soil) (Fig. 2). For both soil depths, bulk OC content was significantly increased by compost (control = low-rate < high-rate) and biochar (control < low-rate < high-rate for surface soil and control < low-rate = high-rate for subsurface soil; Fig. 2, Table S4). Compared to the control, the bulk soil OC content increased at a maximum by 48 %

composting (yes/no), rate (high/low), and their interaction, as well as row and column in the field due to the row-column design for the four mixed treatments: C20B9 (no co-composting), C20B9 (co-composted), C70B31.5 (no co-composting), C70B31.5 (co-composted). Model simplification and residual inspection was carried out as described above, except that a varying of the order of the factors was not required due to the balanced design. The following treatments were missing one replicate for the specified parameter of interest: C20B9 POM OC content for the 10−30 cm depth, B9 WHC for the 0−10 cm depth, C20B9 POM B/A*OC for the 10−30 cm depth, B9 < 0.053 mm B/A*OC for the 0−10 cm depth, and C70 < 0.053 mm B/A*OC for the 10−30 cm depth. In addition, one high outlier of the B9 treatment for the < 0.053 mm B/A*OC was removed due to implausibility of the value, which might have been caused by contamination of the sample during laboratory routine. Removal of this outlier resulted in detection of a significant biochar effect that was not observed with inclusion of the outlier. 3. Results 3.1. Bulk soil properties The effect of the factors compost and biochar on bulk soil properties were analyzed for the treatments Control, C20, C70, B9, B31.5, C20B9 (no co-composting), and C70B31.5 (no co-composting). The WHC ranged from 37.0 % (C20B9) to 42.1 % (B31.5) in the surface soil and 34.3 % (C20B9) to 39.8 % (C70B31.5) in the subsurface soil (Fig. 1, Table S1). We

Fig. 1. Boxplots of bulk soil properties with p values indicating significant treatment effects found by analysis of variance (ANOVA). WHC = water-holding capacity; Cmic = microbial biomass carbon; CEC = cation exchange capacity; C20 = 20 tons compost ha−1; C70 = 70 tons compost ha−1; B9 = 9 tons biochar ha−1; B31.5 = 31.5 tons biochar ha−1. Cmic for both soil depths Box-Cox transformation was required prior to ANOVA. 4

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Fig. 2. Boxplots of soil organic carbon (SOC) content of bulk soil and physical soil fractions with p values indicating significant treatment effects found by analysis of variance (ANOVA). POM = particulate organic matter; C20 = 20 tons compost ha−1; C70 = 70 tons compost ha−1; B9 = 9 tons biochar ha−1; B31.5 = 31.5 tons biochar ha−1. The following parameters required Box-Cox transformation prior to ANOVA: 10-30 cm bulk soil, POM SOC content, and 0-10 cm 2-0.25 mm fraction SOC content.

We did not observe treatment effects on the mass distribution of aggregate size fractions < 2 mm, however, the 10−2 mm fraction was significantly higher in the 0−10 cm soil depth due to application rate, and in the 10−30 cm fraction due to compost application (Figure S1). The surface soil treatments receiving the high biochar application rate (B31.5, C70B31.5) resulted in POM OC storage approximately 10 times higher than POM OC storage in the control (7.2–7.7 versus 0.70 g OC kg soil−1; Fig. 2). Treatments receiving the low biochar application rate (B9, C20B9) showed intermediate increases in OC storage for POM (2.9–3.0 g OC kg soil−1) that were higher than the compost only treatments (C20, C70, 1.1–1.2 g OC kg soil−1). The subsurface POM showed lower OC storage than the surface POM (Fig. 2), but was similar in that the high-rate biochar treatments (B31.5, C70B31.5) had the highest OC storage (3.8–6.3 g OC kg soil−1), the control had the lowest OC storage (0.20 g OC kg soil−1), and the low-rate biochar application resulted in storage of OC between the extremes (1.1–1.3 g OC kg soil−1). The factor biochar significantly increased storage of OC in the 2 -

in surface soil and by 71 % in subsurface soil with the high-rate compost-biochar mixture. For the mixed treatments C20B9, C20B9 (co-composted), C70B31.5, and C70B31.5 (co-composted), we detected a significant increase in all bulk soil properties in response to the higher application rate in both soil depths, except for WHC, which only significantly increased with a higher application rate in the surface soil (Table S3). The factor cocomposting did not significantly affect any measured bulk properties. 3.2. Organic carbon content of particulate organic matter (POM) and aggregate size fractions Biochar significantly increased the storage of OC in POM fraction (control < low-rate < high-rate for the surface; control < low-rate = high-rate for the subsurface), while compost only significantly increased POM fraction OC storage in the subsurface soil (control < high-rate; Fig. 2, Table S4). Aggregate yields (mass proportion of aggregate fractions as a percentage of bulk soil < 10 mm) are shown in figure S1. 5

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depth to represent the fractions that are theoretically the quickest (POM) and slowest (< 0.053 mm) to incorporate new OC. For the surface soil POM, all treatments that contained biochar had orders of magnitude higher B/A*OC than the control or compost only treatments (Fig. 3). The B/A*OC for surface soil POM treatments following high-rate biochar application were 62–78 times higher than the control, while low-rate biochar showed an increase of 22–29 times, and compost only treatments were of similar magnitude to the control, showing an increase by a factor of 2.3 - 3.4. Subsurface soil POM B/ A*OC were lower than surface values, with the exception of the C70B31.5 treatment, and showed a similar pattern of response to treatment as the surface soil (Fig. 3). Subsurface POM B/A*OC was 88–244 times higher for high-rate biochar treatments compared to the control, while lowrate biochar was 26–47 times higher than the control, and the compost only treatments 2.1–3.7 times higher. A significant interaction effect of the factors biochar and compost was found for the surface POM B/A*OC, while in the subsurface, both biochar and compost individually significantly increased POM B/A*OC (biochar: control < low-rate < highrate; compost: control < high-rate; Fig. 3, Table S6). The B/A*OC was less variable and of smaller magnitude for the < 0.053 mm fraction compared to the POM fraction. Biochar significantly increased the < 0.053 mm fraction B/A*OC for the surface (control < low-rate = high-rate) and subsurface soil (control = low-rate < highrate; Fig. 3, Table S6). Surface soil B/A*OC for the < 0.053 mm fraction was 1.3–1.6 times greater for treatments receiving high-rate biochar additions compared to the control, low-rate biochar increased by 1.3 times, and compost only treatments were similar or lower than the control (0.9–1.0 times the control) (Fig. 3). Subsurface soil B/A*OC for the < 0.053 mm fraction was 1.2–1.3 times greater for high-rate biochar treatments compared to the control, while low-rate biochar increased by 1.0–1.4 times, and treatments receiving compost only were lower than the control by a factor of 0.8–0.9. For the mixed compost-biochar treatments, we detected a significant increase in B/A*OC of the POM fraction for both soil depths with the higher rate of treatment application (Table S6). For B/A*OC of the < 0.053 mm aggregate fraction, there was only a significant increase in response to the higher rate of treatment application in the subsurface soil. The factor co-composting did not show any significant effect on B/A*OC contents.

0.25 mm fraction for the surface and subsurface soil (control < highrate), while the factor compost only significantly increased OC storage in the 2 - 0.25 mm fraction for the subsurface soil (control = lowrate < high-rate; Fig. 2, Table S4). The surface and subsurface soil 2 0.250 mm fraction had the greatest OC storage for the high-rate compost-biochar mixture (16.2 g OC kg soil−1 for surface and 13.1 g OC kg soil−1 for subsurface) compared to the other treatments (9.6–12.4 g OC kg soil−1 and 8.4–10.7 g OC kg soil−1 for surface and subsurface, respectively) (Fig. 2). Contributions of the 0.250 - 0.053 mm fraction to the OC content of bulk soil was similar among all treatments for the surface (3.1–4.1 g OC kg soil−1) and subsurface (3.1–4.4 g OC kg soil−1) depths (Fig. 2). Biochar significantly increased OC storage in this fraction for both soil depths (control < high-rate), whereas compost only significantly increased subsurface OC storage in this fraction (control < high-rate; Fig. 2, Table S4). Storage of OC in the surface (0.7–1.0 g OC kg soil−1) and subsurface (0.6–0.9 g OC kg soil−1) < 0.053 mm fraction were also similar (Fig. 2). The factor biochar significantly increased the OC storage in this fraction (control < low-rate = high-rate for the surface soil; control < high-rate in the subsurface soil), while compost had no effect (Fig. 2, Table S4). The surface soil POM fraction accounted for an average of 15.1 % of the bulk soil OC; however, the storage of OC in POM varied widely among treatments, from 3.8 % (control) to 30.3 % (B31.5) of the bulk OC content (Fig. 2). The remaining surface fractions contained a more consistent allocation of bulk OC across treatments, with 53.2 %, 16.5 %, and 3.8 % of the bulk OC content held on average in the 2 - 0.250 mm, 0.250 - 0.053 mm, and < 0.053 mm fraction, respectively. In the subsurface soil, the POM fraction accounted for an average of 10.8 % of the bulk soil OC, the 2 - 0.250 mm fraction accounted for 58 %, the 0.250 0.053 mm fraction for 20 % and the < 0.053 mm fraction for 4.0 % of the SOC. For the mixed compost-biochar treatments, we only detected a significant increase in OC contribution of POM and the 2 - 0.250 mm aggregate fraction to bulk soil OC content due to the higher rate of treatment application (Table S5). The factor co-composting did not show any significant effect on POM or aggregate fraction OC storage. 3.3. Composition of organic matter as analyzed with infrared spectroscopy

4. Discussion The B/A ratio derived from the DRIFT spectra was multiplied by the OC storage (g OC kg soil−1) of each sample to obtain a semi-quantitative measure of the OM functional composition in terms of the relative amount of C]O groups to aliphatic C–H groups (Fig. 3). We only measured DRIFT for POM and the < 0.053 mm fraction for each soil

4.1. Bulk soil properties Recent studies focused on effects of biochar addition to poor quality soils (Gaskin et al., 2010; Glaser et al., 2015; Liu et al., 2012a, 2013; Fig. 3. Boxplots of B/A*OC for particulate organic matter (POM) and < 0.053 mm fraction with p values indicating significant treatment effects found by analysis of variance (ANOVA). POM B/A*OC for both soil depths required Box-Cox transformation prior to ANOVA. C20 = 20 tons compost ha−1; C70 = 70 tons compost ha−1; B9 = 9 tons biochar ha−1; B31.5 = 31.5 tons biochar ha−1. “A” represents the “aliphatic” (C-H) of the Diffuse Reflectance Infrared Fourier Transform Spectroscopy, while “B” represents the “carbonyl” and “carboxyl” (C = O) region.

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also aided with breakdown and incorporation of added OC into POM and aggregates. When we look at the pairwise comparisons, it is apparent that often the low biochar application rate (9 t ha−1) was sufficient to significantly increase OC storage in bulk and fraction OC compared to the control, and the high-rate application (31.5 t ha−1) always significantly increased bulk and fraction OC content in both soil depths compared to the control six years after application. This corroborates the long-term C sequestration potential of biochar even under practical agricultural field conditions. Compost, however, only significantly increased bulk and fraction OC at the high application rate (70 t ha−1) compared to the control, but not at the low application rate (20 t ha−1). This suggests that, with more time, the influence of compost on bulk OC storage may decrease because of more rapid compost decomposition, confirming long-term field observations (Jenkinson, 1991). Differences in POM−OC were mainly driven by biochar application; however, compost also significantly increased POM−OC storage in the subsurface soil. This confirms results from Grunwald et al. (2017), who analyzed samples from a field experiment in Germany one year after 30 t ha−1 of biochar were mixed into the first 20 cm of a silty loam soil. In this study, more than 80 % of the total biochar-C was found in the free light fraction, which plays a similar role ecologically to the separated POM in this study. It has to be considered that the observed increase in the POM yield and OC storage due to biochar particles will most likely lead to a change in the soil ecological function of this fraction. In soils not receiving biochar additions or additions from not pyrolyzed organic material, the POM fraction (i.e. free organic particles) represents the soil OM compartment turning over faster than most of the other operationally-defined fractions separated from soil (Gregorich et al., 2006). The mean residence time of this fraction will most likely distinctly increase with an increased proportion of biochar and the POM fraction might react much slower to changes in management and environmental conditions than it does without significant contribution from biochar. For all aggregate size fractions (2 - 0.250, 0.250 - 0.053, and < 0.053 mm), biochar significantly increased OC storage in the surface soil after six years, while both biochar and compost significantly increased subsurface OC storage in all aggregate fractions, with the exception of the < 0.053 mm fraction, which was not affected by compost application. These results are in contrast to those from Grunwald et al. (2017), who did not find differences in aggregate OC dynamics one year after addition of 30 t ha-1− biochar to a temperate climate soil with 91 % of clay + silt and 1.2 % OC. The soil in the present field experiment (clay + silt: 38 %, soil OC: 1.4–1.9 %) and Grunwald et al. (2017) both have relatively good initial soil structure derived from inorganic and organic binding agents . The difference in results between these studies point to time-dependent changes (Hagemann et al., 2017a; and 2017b), which may affect biochar characteristics and its ability to promote aggregate formation. It also has to be kept in mind that biochar incorporation into aggregates is highly affected by particle size and time for weathering, with aggregation potential increasing with decreasing biochar particle size (Zhang et al., 2015), and may be influence by biological effects, such as earthworm activity. However, comparative data on the size distribution of the added biochar between this study and Grunwald et al. (2017) are not available and studies analyzing the effect of biochar particle size on soil and aggregate OC dynamics under field conditions are scarce.

Major et al., 2010). In contrast, our soil exhibited better initial conditions (WHC = 38 %, bulk soil OC = 1.8 %) compared to others (e.g., Liu et al., 2013; WHC = 6–18 %, bulk soil OC = 0.8 %). There was a lack of consistent effects on WHC in this study, with a significant interaction effect between compost and biochar in the surface soil preventing interpretation of the individual effects of these treatments. There was only a significantly higher WHC in the subsurface soil for the high-rate compost treatment compared to the low-rate treatment, with no significant difference observed by comparing to the control. These unclear results may be attributed to the high inherent WHC of the soil used in this study and the given field variability, which may have prevented detection of treatment-derived changes. Unlike WHC, we did observe distinctly higher soil Cmic contents in the surface and subsurface soils in response to the C70B31.5 treatment compared to the control; however, only the subsurface soil showed a significantly higher Cmic following high-rate compost application compared to control. This result may be due to the more limited microbial communities present at greater soil depth, and therefore the subsurface soil was more sensitive to influxes of OC. The data suggest that especially in the surface soil, field variability might be too high to detect the effects of even high rates of OC application on Cmic. In contrast, the subsurface soil is less affected by environmental conditions such as wetting and drying, which are known to affect the microbial community structure (Kaiser et al., 2015), leading to less variability in the field and stronger management effects on Cmic. The positive effect of compost on subsurface soil Cmic may have greater implications for promoting soil microbial communities deeper in the soil at the plant root zone. Six years after biochar and compost application at high rates, the combination of habitat (biochar) and substrate (compost) provides the most favorable conditions for microorganisms, suggesting a longerlasting, more sustainable effect of this management option. A shift towards more favorable conditions for soil microorganisms and, therefore, increased Cmic contents in soils that received biochar additions is often closely linked to an increase in soil pH (Luo et al., 2013). We did observe a link between Cmic and pH, with the greatest pH increase in this slightly acidic soil observed in the high-rate biocharcompost mixtures at both soil depths, which also produced the highest Cmic. While high-rate biochar application significantly increased pH in the surface soil compared to the control, high-rate compost significantly increased pH in both soil depths compared to the control, suggesting compost was of greater influence overall. It can be assumed that after biochar addition, the pH rose and then declined until sampling, which was observed in a three-year field experiment under temperate climate by Jones et al. (2012). The authors found the highest soil pH-values in year two, followed by a decline. After three years, the soil containing “aged” biochar showed pH-values that were two units lower than soil containing “fresh” biochar, which is a trend confirmed by other studies (Castaldi et al., 2011; Mukherjee et al., 2014). This suggests that under biochar addition, favorable pH conditions for microorganisms are more distinct in the first two years (Kolb et al., 2009; Luo et al., 2013). The strongest increase in soil CEC in the surface and subsurface soils compared to the control was for the high application rate of the compost-biochar mixture. Immediate and longer-lasting positive effects of biochar applications on soil CEC are generally confirmed by other studies (e.g., Cheng et al., 2008; Gaskin et al., 2010; Zhang et al., 2016). However, six years after application, the driver for the increased CEC in this field experiment was high-rate compost application. Cation exchange capacity and pH are tightly linked; therefore, it is not surprising that the same factor, i.e. reactive compost, had a stronger influence on both properties than biochar.

4.3. Composition of organic matter as analyzed with infrared spectroscopy The POM fraction was expected to be the quickest to incorporate added OC, and did show improved functional composition (B/A*OC) in response to biochar application, representing an increase in the overall reactivity of this fraction as well as its contribution to cation exchange processes (Kaiser et al., 2008). The increase in B/A*OC for POM was proportional to the rate of biochar addition (B31.5 > B9 > B0). The OM

4.2. Organic carbon content in bulk soil, particulate organic matter, and aggregate size fractions At both depths, biochar and compost had a significant role in increasing bulk soil OC contents. Annual shallow tillage of plots may have 7

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application of 31.5 t biochar ha−1. Biochar addition may lead to a lower mean turnover rate in the POM fraction, which can shift the soil ecological relevance of this fraction from one composed of plant-derived organic particles to thermally-altered plant particles storing more OC for a longer time. Positive effects of biochar and compost application on the OC storage in aggregate size classes gave further insight into possible treatment effects on OC storage and aggregate dynamics. Only the application of biochar promoted OC storage in aggregates in the surface soil, while both biochar and compost were drivers of aggregate formation in the subsurface soil. Lower applications did not always result in significant treatments effects six years after application, suggesting that more frequent or higher rates of application are required to see benefits of organic amendments.

associated with the < 0.053 mm fraction is generally more strongly protected against decomposition by occlusion in aggregates than the OM recovered with the POM fraction (Poeplau et al., 2018), and is, therefore, expected to incorporate added OM at a slower rate. After six years, both soil depths demonstrated significant differences for B/A*OC in POM due to both biochar and compost application, while the < 0.053 mm fraction was only significantly affected by biochar at the high and low rates of application for the surface soil, and at only the high-rate application in the subsurface. This point towards a change in the quality of OM stored in protective aggregates < 0.053 mm in response to the biochar application. The increase in reactive C]O groups (B/A*OC) in this fraction might lead to enhanced interactions between OM and minerals, potentially leading to an increased importance of this fraction for the long-term stabilization of OM. The increase in absorption intensities at around 1600 cm−1 resulting in elevated B/A*OC values might also have been a result from a biochar derived increased in aromatic moieties (Glaser et al., 2015; Wiedner et al., 2015). However, in this study the DRIFT spectra did not show further absorption maxima at two other wave lengths (1500 cm and 1580 cm−1), which are characteristic for aromatic moieties (Hesse et al., 2012). Based on that we interpret the increase in the B/A*OC data in this study as a result from microbially mediated oxidation of biochar surfaces occurred during the six years of biochar aging under field conditions (Joseph et al., 2010).

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest None. Acknowledgements

4.4. Effect of co-composting on biochar-compost mixtures We would like to thank Anja Sawallisch from the University of Kassel, Department of Environmental Chemistry for technical assistance. We also want to acknowledge the support of the Landwirtschaftliche Lehranstalten Bayreuthby providing the field area for experimentation and Martin Höpfel for technical support.

For the mixed treatments C20B9 (low-rate) and C70B31.5 (high-rate) that were either co-composted or mixed just prior to application, we detected a significant increase in bulk OC content, POM−OC, 2 - 0.25 mm−OC, CEC, Cmic, pH, and POM B/A*OC in both soil depths, and WHC and < 0.053 mm B/A*OC in the subsurface soil in response to the high application rate compared to the low application rate. The selective increase of POM−OC and 2 - 0.25 mm−OC due to higher application rate points toward a time dependent density (POM-C) and particle size (macro-aggregates > 0.25 mm) effect of the added material and the promotion of macro-aggregate formation for multiple years only if a certain amount of additional binding agents is exceeded. After six years of application biochar-compost mixtures at the low rate, it seems that the percentage of processed and size degraded material was too high to have an effect on POM-C and 2 - 0.25 mm−OC. However, the factor co-composting was not significant for any of the measured soil parameters. Hagemann et al. (2017a) observed that co-composting biochar and compost reduced overall surface reactivity and binding agents for aggregates such as microbial biomass by locking up pores through interactions between the biochar surface and compost products, however an opposing trend was observed by Wiedner et al. (2015). These conflicting results point to unidentified mechanisms shifting biochar co-composting between a negative and positive factor, such as soil properties, climate, length of time composted, or amount of aging in the soil, indicating that further research is needed. Our data suggest that, for the analyzed soil environment, soil ecological benefits are less related to the process of co-composting and more influenced by the amount of biochar-compost mixture added to soil, which might be helpful in designing specific sustainable management strategies.

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