Biochar and its combination with nitrogen fertilization altered soil organic matter, humic substances, and soil structure: Short-term vs. long-term changes

Biochar and its combination with nitrogen fertilization altered soil organic matter, humic substances, and soil structure: Short-term vs. long-term changes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biochar and its combination with nitrogen fertilization altered soil organic matter, humic substances, and soil structure: Short-term vs. long-term changes Vladimír Šimanský, Elżbieta Wójcik-Gront, Sanandam Bordoloi, Ján Horák This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7091756/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in Environmental Geochemistry and Health → Version 1 posted 7 You are reading this latest preprint version Abstract Biochar (B), as well as its combination with nitrogen (N) fertilization, can influence soil quality and fertility. Humus formation and aggregation is a long-term process in soils and the impact of combined biochar-N fertilization on its formation remains underexplored for long term studies. The aim of this study was to determine the extent to which combined biochar-N fertilization on the soil organic matter (SOM) content, quality of humic substances (HS), and soil structure. We also aimed at quantifying changes in the relationships between HS and soil structure. Silty loam Haplic Luvisol was sampled from the field after 1- and 9- years from the incorporation of biochar (0, 10, and 20 t ha − 1 of biochar marked as B0, B10, B20) combined with N fertilization (N0, N1, and N2). The results showed that B + N fertilization moderately increased the soil organic carbon (Corg) content in the soil after 1 year of incorporation. After 9 years, the Corg content in the soil was relatively balanced among the treatments. Only in B20N2 did the HS content significantly increase compared to B0N0. In B20N2, the content of microaggregates significantly decreased compared to B0N0 after 9 years. Significant changes in correlations between SOM, HS, and aggregate size fractions suggest potential shifts in their relationships over the decade. The gradual strengthening and changes in the intensity of positive or negative relationships between them suggests the aging of biochar may have long term effects on crop productivity and soil health. biochar fulvic acids humic substances soil organic matter soil aggregates Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Multiple perspectives and approaches have existed regarding soil structure and humic substances in agricultural soils, with some being more or less preferred by soil scientists. For example, Amezketa ( 1999 ) and Bronick and Lal ( 2005 ), in their reviews on soil structure, addressed a wide range of environmental and anthropogenic factors and their interactions, emphasizing soil aggregates and their stability. Numerous recent scientific studies on soil structure (aggregate stability and its measurement) follow this direction (Šimanský, 2014 ; Memedov et al., 2021; Šimanský et al., 2023 ; Guo et al., 2024 ; Siebers et al., 2024 ). On the other hand, Levy (1991) presented a different perspective on soil structure, focusing on the arrangement of soil pores and highlighting the importance of their unique architecture with time. Regardless, soil structure is a fundamental physical property that deserves attention (Foth, 1990 ; Lal and Shukla, 2004 ; Blume et al., 2016 ). Similarly, the study of humus in soil, despite being a subject of scientific inquiry for over 200 years, has seen various perspectives and approaches (Stevenson, 1994 ; Lehmann and Kleber, 2015 ). The most crucial aspect is that humus is a component of soil organic matter (Stevenson, 1994 ; Weil and Brady, 2017 ), playing critical roles in soil formation and its physical, nutritional, chemical, and biological properties (Poláková et al., 2018; Mu et al., 2024 ). Literature corroborates that soil structure largely results from the content and quality of humic substances in the soil (Poláková et al., 2018). An existing knowledge gaps have been identified concerning the relationships between soil structure and humic substances following the application of biochar, biochar substrates, or their combinations with mineral fertilization (Zhang et al., 2014 ; Mierzwa-Hersztek et al., 2018 ; Juriga et al., 2019 ). Biochar generally refers to a type of porous, highly aromatic, insoluble substance produced by the pyrolysis of biomass, such as lignocellulosic biomass and plant waste, under low-oxygen or anoxic conditions (Hansen et al., 2016 ). Biochar has a high content of stable carbon, which resists decomposition and remains in the soil for a long time, contributing to carbon sequestration (Hossain et al., 2020 ; Šrank and Šimanský, 2020 ). Due to its high specific surface area, micropore content (Chintala et al., 2014 ), lower bulk density (Glab et al., 2016), and the presence of functional groups such as phenolic hydroxyl groups, alcoholic hydroxyl groups, and carbonyl groups, etc. (Xu et al., 2014 ; Glab et al., 2016), as well as other benefits (regulation and immobilization of harmful substances such as heavy metals – Shen, 2024 ), biochar can be applied to soil as an additive that manages water regime (Igaz et al., 2018 ; Sharma, 2024 ), nutrient regime (Chen et al., 2018 ; Li et al., 2020 ), cation exchange capacity and soil sorption properties (Igaz et al., 2018 ; Hossain et al., 2020 ), and reduces soil bulk density (Glab et al., 2016). From soil physical properties, biochar applied to soil can influence soil structure (Juriga and Šimanský, 2018 ) by improving soil aggregation and creating a more stable and porous soil matrix (Zhang et al., 2022 ). Biochar also enhances soil structure by reducing soil compaction, especially in texturally heavy soils with higher clay content (Blanco-Canqui, 2021 ). The resulting favorable soil structure provides an excellent foundation for root growth and microbial activity, potentially benefiting crop performance through higher crop yields. Biochar, as well as its combination with nitrogen (N) fertilization, can enhance soil properties, thereby improving the quality and fertility of arable land. We have hypothesized three scenarios on how it may enhance that. First, since humus formation is a long-term process, the effect of biochar and its combination with N fertilization on humus formation will be more pronounced after prolonged residence in the soil (H1). Second, assuming that the prolonged presence of biochar in the soil would improve soil structure by reducing microaggregates and increasing mainly favorable soil macroaggregates in a size fraction of 0.5-3 mm over time (H2). Third, changes in soil organic matter and humic substances may have a direct impact on the size fractions of soil aggregates (H3). Based on the context and hypotheses, the objectives of this study were (i) to determine the extent to which biochar and its combination with N fertilization on the content and quality of humic substances and soil structure and (ii) to quantify changes in the relationships between humic substances and soil structure. 2. Material and methods 2.1. Site description The Slovak University of Agriculture's experimental site in Nitra (Dolná Malanta) is located at 48°10'00" N, 18°09'00" E, in the northeastern Danubian Lowland and the western Žitava Upland, near the lower basin of the Selenec stream. This site is 4 km east of Nitra, within a warm maize-growing region. The experimental area features flat terrain with a gentle southwest slope at an elevation of 170–180 meters above sea level. Geologically, it sits at the boundary between the crystalline-Mesozoic massif of the Tribeč Mountains and the Žitava Upland. The geological base is primarily composed of eluvial-deluvial sediments from the Tribeč Mountains, mixed with loess sediments from the Žitava Upland in some areas. The soil type is Haplic Luvisol, with a silt loam texture in the A-horizon. The soil contains an average of 360.4 g kg − 1 sand, 488.3 g kg − 1 silt, and 151.3 g kg − 1 clay and has a slightly acidic pH of 5.71. Before the experiment began, the soil had low organic carbon content (9.13 g kg − 1 ), a moderately low cation exchange capacity (142 mmol p + kg − 1 ), and a saturated sorption complex (85%). The site is in a very warm and dry agro-climatic region, with an average annual precipitation of 559 mm and an average annual temperature of 10.7°C (based on data from 1991–2020). 2.2. Experimental design The biochar experiment was established in 2014, prior to the sowing of spring barley, and is ongoing. The crop rotation was as follows: 2014 - spring barley, 2015 - maize, 2016 - spring wheat, 2017 - maize, 2018 - spring barley, 2019 - maize, 2020 - peas, 2021 - winter wheat, 2022 - maize, and in 2023 - spring barley. The original experiment consists of 9 treatments detailed in Table 1 . The experiment was set up using a randomized block design with three replications for each treatment. Each plot measured 6 x 4 m and was separated by a 1 m protective strip. Biochar was applied to the soil surface in the spring of 2014 at the respective treatment rate, manually spread over each plot, and then incorporated into the soil to a depth of 10 cm using a combinator. Biochar was applied at rates of 10 and 20 t ha − 1 (B10 and B20). The first level of nitrogen fertilization (N1) was determined based on the requirements of the specific crop grown in that year and calculated as per practices in the Slovak Republic. The second level of nitrogen fertilization (N2) was increased by 100%. Throughout the experiment, nitrogen fertilization rates ranged from 30 to 160 kg N ha − 1 for N1 and from 45 to 240 kg N ha − 1 for N2. Specifically, in 2015, the rates were 160 and 240 kg ha − 1 for N1 and N2, respectively, and in 2023, the rates were 40 kg ha − 1 for N1 and 80 kg ha − 1 for N2. Nitrogen fertilization was always carried out during the growing season of the cultivated crop, with the nitrogen doses divided into 2–3 applications depending on the amount of nitrogen. The soil was not plowed during the experiment; it was only disked or surface-tilled to a maximum depth of 15 cm. Standard agronomic practices during the growing season of the cultivated crops were carried out each year (including disease, pest control, and weeding). Table 1 Experimental design for the current study Sl. No. Designation Description 1 B0N0 no biochar, no nitrogen 2 B10N0 biochar at a rate of 10 t ha − 1 and no nitrogen 3 B20N0 biochar at a rate of 20 t ha − 1 and no nitrogen 4 B10N1 biochar at a rate of 10 t ha − 1 and a first level of N fertilization 5 B20N1 biochar at a rate of 20 t ha − 1 and a first level of N fertilization 6 B10N2 biochar at a rate of 10 t ha − 1 and a second level of N fertilization 7 B20N2 biochar at a rate of 20 t ha − 1 and a second level of N fertilization 2.3. Biochar and nitrogen mineral fertilizers The applied biochar was produced from cereal husks and waste sludge generated during paper production. The pyrolysis temperature was 550°C with a duration of 30 minutes. The biochar contained 53.1% total carbon, 1.4% total nitrogen, 57 g kg − 1 calcium, 3.9 g kg − 1 magnesium, 15 g kg − 1 potassium, 0.7 g kg − 1 sodium, and 38.3% ash. The specific surface area was 21.7 m 2 g − 1 . Its pH was 8.8, and the particle size of the produced biochar ranged from 1 to 5 mm. Nitrogen was applied in the form of the following commercial mineral fertilizers: LAD27, DASA 26/13, and NPK. 2.4. Soil sampling and analysis For the purposes of sampling in this study, soil samples were collected from a depth of 20 cm from each replication of the respective treatment at monthly intervals from April to September in 2015 and from April to July in 2023. Soil samples were taken to determine the size fractions of soil aggregates and parameters of soil organic matter (SOM). The soil samples were carefully collected using a spade to minimize damage to the soil aggregates. After drying, the samples were sieved through a set of sieves 3, 0.5, and 0.25 mm. The aggregates remaining on the sieve were then weighed and expressed as a percentage of the total soil weight. The following size fractions of dry soil aggregates (DSA) were obtained: >3 mm, 3-0.5 mm, 0.5 − 0.25 mm, and < 0.25 mm. The size threshold separating macroaggregates (ma) from microaggregates (mi) is 0.25 mm. Agronomically valuable macroaggregates are in the 0.5-3 mm range (Fulajtár, 2006 ; Šimanský et al., 2023 a). Soil samples for determining SOM and humus were homogenized, ground, sieved through a 0.25 mm sieve, and then analyzed using standard methods. Soil organic carbon (Corg) was quantified using the wet combustion technique, which involves the oxidation of soil organic matter (SOM) with a mixture of 0.07 mol L − 1 H 2 SO 4 and K 2 Cr 2 O 7 , followed by titration with Mohr’s salt (Dzadowiec and Gonet, 1999a). The group and fraction composition of humic substances (HSs) including humic acids (HAs) and fulvic acids (FAs) were analyzed using the method described by Belchikova and Kononova, which includes extraction with a mixture of 0.01 mol L − 1 Na 4 P 2 O 7 , 10 H 2 O, and 0.1 mol L − 1 NaOH (Dzadowiec and Gonet, 1999b). The light absorbance of HSs and HAs at wavelengths of 465 and 650 nm was measured using a Jenway 6400 Spectrophotometer to determine the color quotients of humic substances (Q 4/6 HS ) and humic acids (Q 4/6 HA ). 2.5. Statistical analysis First, a two-way ANOVA was applied to determine whether year and treatment significantly influenced the observed variables. Subsequently, sampling time (April-September for 2015 and April-July for 2023) was treated as a repetition in the one-way ANOVA conducted separately for each year, with treatment as the factor. Analysing each year separately avoids the assumption that treatments had identical effects in both years, allowing for a more nuanced understanding of treatment impacts. Pearson’s correlation analysis was performed to examine relationships between different measured variables within each treatment for 2015 and 2023 separately. Principal Component Analysis (PCA) was conducted to reduce the dimensionality of the dataset while preserving the most significant variance. The first two principal components (PC1 and PC2) were selected for visualization and interpretation, as they accounted for the largest proportion of variance. A biplot was used to illustrate the relationships between treatments and variables, highlighting major patterns in the dataset and demonstrating how experimental treatments affected variable relationships and overall variance. 3. Results and discussion 3.1. Effect of biochar on soil organic matter, humic substances, and soil structure The Corg content was generally higher in 2023 than in 2015 (Fig. 1), indicating a moderate improvement in soil management practices and aligning with current global challenges such as sustainable agriculture, carbon neutrality (Lal, 2004 ; Rodrigues et al., 2023 ). The Corg content in B0N0 (control treatment) increased from 1.22% in 2015 to 1.51% in 2023. Soil management alternates Corg in soils (Rodrigues et al., 2023 ), leading to various scenarios ranging from negative through neutral and stable to positive (Weil and Brady, 2017 ). Overall, the positive scenario in this study was attributed to several factors, such as no-tillage and reduced tillage intensity (Kan et al., 2022 ), leaving crop residues on the soil surface (Ahmad et al., 2024 ), and the application of biochar (Šrank and Šimanský, 2020 ; Li et al., 2024 ). The effects of biochar application and its combination with N fertilization on changes in Corg content differed between 2015 and 2023. In 2015, Corg content increased by 32%, 21%, 51%, 31%, and 34% in B20N0, B10N1, B20N1, B10N2, and B20N2, respectively, compared to B0N0. It appears that biochar application increases soil C as a result of the negative priming effect of SOM by biochar and its combination with N fertilization (Kalu et al., 2024 ). Biochar is a source of stable C (Meng et al., 2024 ), and its incorporation into the soil can reduce SOM mineralization. However, labile C, formed during SOM decomposition by microbial activity, can be sorbed onto biochar (Jones et al., 2012 ), thus increasing Corg content as documented by the results of this study (Fig. 1). In this study, a higher rate of biochar was more effective than the lower rate in increasing Corg. Adequate nitrogen is essential for biological processes and supporting microbial activity. Biochar can contain nutrients, including nitrogen (Ippolito et al., 2015 ), which can influence the mineralization of SOM and biochar particles in the soil. Corg content was relatively balanced among treatments in 2023, suggesting that properly set soil management was more decisive than the persistent effect of biochar and its combination with N fertilization. The suppressed effect of biochar on Corg content could be due to its stability and reduced surface activity, as biochar particles were part of soil aggregates (Šimanský, 2016 ) and soil casts after increased earthworm activity (Šimanský et al., 2019 ). In 2015, the extraction of humic substances in SOM (extHS) decreased with the subsequent increase in Corg, indicating reduced mineralization and humification of SOM and the subsequent accumulation of Corg in the soil due to the negative priming effect (Kalu et al., 2024 ). The extraction of humic acids in SOM (extHA) as part of HSs significantly decreased only in B20N1, B10N2, and B20N2 treatments. The extraction of fulvic acids in SOM (extFA) significantly decreased only because of the application of a higher rate of biochar and its combination with the first level of N fertilization in 2015. In 2023, extHS, including extHA and extFA, did not change depending on the treatment (Fig. 1). These results indicate that while biochar increased the content of Corg in the soil, it did not affect the extraction of HSs in SOM, confirming that biochar is characterized by lower reactivity and a more stable structure (Nguyen et al., 2010 ; Gupta and Germida, 2015 ). The degree of humification of organic matter after the application of biochar and its combination with N ranged from 15–24% and from 20–24%, corresponding to a low to medium degree of humification in 2015 and 2023, respectively. It is evident that primary organic matter predominates over humic substances in the soil under treatments, which generally mineralizes more and humifies significantly less (Váchalová et al., 2016 ). Based on the obtained results, it is evident that the application of biochar and its combination with N fertilization did not clearly enhance the formation of humic substances in SOM; rather, it reduced humification. H1 was partially supported because, in 2015, a reduction in humification was observed under all treatments with biochar and its combinations with nitrogen fertilization. After 9 years, there was a trend of increasing extraction of HS and HA in SOM under treatments B10N0, B10N1, B10N2, and B20N2. The utilization of various types of biochar has great potential for the formation and stabilization of humic substances (Li et al., 2015 ; Mierzwa-Hersztek et al., 2018 ), which was partially supported in this study (Fig. 2). Through biochar and its combination with N fertilization, humic substances were not added to the soil. No significant differences were observed between 2015 and 2023, i.e., 1 and 9 years after biochar application. A moderate increase in C HS was found only in 2023 after the application of 20 t of biochar ha⁻¹ and an increased level of N fertilization (N2). In other treatments, an insignificant trend in the increase of C HS was observed. The C HA :C FA ratio in all treatments, including the control, was above 1, indicating overall favorable humus quality in the soil at the experimental field. The C HA :C HA ratio did not change significantly in any year. Nevertheless, an insignificant trend of improving the C HA :C FA ratio in favor of C HA was observed after the application of biochar, while a higher level of N fertilization tended to decrease the C HA :C FA ratio. Humus in the soil is relatively stable (Stevenson, 1994 ; Weil and Brady, 2017 ), but due to external influences, such as the application of biochar in this study, its stability can be disrupted, and soil humus begins to be attacked by soil microorganisms (Adani et al., 2007 ), resulting in a reduction of humic substances in the soil (Mierzwa-Hersztek et al., 2018 ). Of course, the properties of biochar have a crucial impact on these processes (Zhao et al., 2017 ) and is also dependent on the biochar type. In all treatments, the color quotient values of humic substances and humic acids suggested more humified and mature organic matter in the soil, with a high content of condensed aromatic compounds and a low representation of aliphatic compounds (Weil and Brady, 2017 ; Weber, 2020 ). The molecular weight and degree of condensation of humic substances remained unchanged (Fig. 2). Soil structure is fundamentally modified by external factors in addition to internal factors (Amézketa, 1999; Bronick and Lal, 2005 ). Among external influences within agronomic practices, the application of farmyard manure, composts (Šimanský et al., 2021 ), or organic amendments such as biochar (Juriga and Šimanský, 2018 ; Baiamonte et al., 2019 ) is recommended to improve soil structure. Reactive surface functional groups of biochar can form organo-mineral complexes with finer soil particles (clay), which are the basis of soil microaggregates (Brodowski et al., 2006 ). Soil structure formation is mostly associated with microbial activity. Another mechanism is explained through microscopic fungi that can colonize and grow in the pores of biochar, thus supporting the formation and stability of soil structure (Cross et al., 2014 ). The aggregation process is also significantly influenced by the properties of the applied biochar. N fertilization applied with biochar can act as an accelerator, speeding up the mineralization of SOM and biochar particles, improving microbial activity, and resulting in soil aggregate formation (Šimanský, 2016 ). However, soil structure formation in the case of biochar is also the result of external influences, including climatic conditions. In this study, the application of biochar in both rates and its combination with both levels of N fertilization (N1, N2) did not have a statistically significant effect on changes in DSAma content 1 and 9 years after their application. DSAma contents > 3 mm were significantly higher in 2023 than in 2015 (p < 0.001), whereas DSAma contents 0.25-3 mm were significantly higher in 2015 than in 2023 across all treatments (p < 0.001). The results suggest that climatic conditions had a greater impact on soil structure than biochar itself (Fig. 3). Climate, through cycles of soil drying and wetting, as well as freezing and thawing cycles, has a significant effect on soil structure (Foth, 1990 ; Lal and Shukla, 2004 ; Bronick and Lal, 2005 ; Blume et al., 2016 ). In terms of total precipitation and average temperatures, the years 2015 and 2023 were evaluated as moisture-normal, extremely warm and extremely wet, and very warm, respectively, compared to the climatic norm of 1991–2020 (unpublished data). Temperature and moisture changes reflect the activity of soil microorganisms, which have a crucial impact on aggregation and structure formation (Bronick and Lal, 2005 ). DSAmi contents were significantly lower in 2023 than in 2015 (p < 0.001). However, in 2015, biochar and its combinations with N fertilization had no effect on DSAmi, whereas in 2023, i.e., 9 years after its incorporation into the soil, a positive effect was identified. DSAmi contents after the application of 20 t of biochar ha⁻¹ decreased by 33% and 30% compared to B0N0 (control) and B10N0, respectively. This suggested that microaggregates bonded with biochar particles into larger macroaggregate size fractions, which were evenly balanced across treatments (Fig. 3). Overall, the effects on soil structure are varied and tend to manifest after long-term biochar application in the soil rather than in a short time interval after its incorporation (Jien and Wang, 2013), as documented by the findings of this study (Fig. 3). Based on the results of this study, H2 was partially supported because, after the first year, no trends or significant changes in soil aggregate contents were observed under the individual treatments. After 9 year from the establishment of the experiment, it was found that the content of microaggregates was significantly reduced in the B20N2 treatment, and a trend of decreasing microaggregate content was observed in treatments such as B20N0, B10N1, B20N1, and B10N2. However, this did not translate into a substantial increase in agronomically valuable size fractions of macroaggregates (0.5-3 mm). 3.2. Relationships between soil organic matter, humic substances, and soil structure SOM and aggregation are closely related, as SOM influences soil structure stability by acting as a binding agent that connects mineral particles, reduces aggregate wettability, and thus affects their mechanical strength (Onweremadu et al., 2007 ). Soil organic carbon binds with clay particles through polyvalent cations, forming an organo-mineral complex, which is the foundation of soil structure (Bronick and Lal, 2005 ). Humic substances within SOM have a significant effect on soil structure (Polláková et al., 2018 ; Weber, 2020 ), as does the application of biochar and its combination with N fertilization (Juriga et al., 2019 ). As previously mentioned, biochar is a source of stable carbon with lower reactivity and a more stable structure (Nguyen et al., 2010 ; Gupta and Germida, 2015 ; Meng et al., 2024 ). When biochar is applied with readily available N, it can promote SOM mineralization through microorganisms (Ali et al., 2020 ), which produce binding agents responsible for connecting soil particles, resulting in improved soil structure (Costa et al., 2018 ). In this study, the relationships between SOM, humic substances, and aggregate size fractions varied across all treatments depending on the year (Table 2), potentially due to changes in fundamental biological mechanisms. Notably, no statistically significant correlations were found between Corg and individual size fractions of DSAma and DSAmi, either 1 or 9 years after biochar application. This phenomenon is likely related to the quality of SOM. Oades ( 1984 ) stated that not all organic compounds in soil are responsible for aggregation. Different forms of organic matter stabilize aggregates of various sizes and sometimes may not influence soil aggregation at all. For example, anions of organic compounds such as fulvates, citrates, oxalates, or acetates increase clay dispersion, while aromatic acids (salicylic acid, p-hydroxybenzoic acid) have a flocculating effect (Itami and Kyuma, 1995 ). This is partially supported by the findings of this study, where primarily lower contents of extFA in SOM and CFA, because of the effect of biochar applied at a rate of 10 t ha⁻¹ and its combination with both levels of N fertilization after 1 year (Fig. 1), increased the contents of individual size fractions of DSAma and DSAmi (Table 2). The relationships between SOM, humic substances, and soil structure were markedly different depending on the treatment. For example, B10N0 was associated with high values of variables moving in the same direction (DSAma 0.5 − 0.25 mm in 2015, CHA in 2015). This treatment was significantly different from B20N2, where the opposite trends were observed (Fig. 4). Across all treatments, the overall number of significant relationships was higher in 2015 than in 2023 and often differed markedly within treatments (Table 2; Fig. 4). This suggests complex interactions between internal factors (SOM, humic substances, soil structure) and external factors (weather conditions affecting soil temperature and moisture). H3 was partially supported because a significant relationship between the monitored parameters of SOM, humic substances, and size fractions of soil aggregates was not consistently observed across treatments and years. 4. Conclusions The results indicate that the parameters of soil organic matter (SOM), humic substances, and soil structure, as well as their long-term interrelationships, significantly varied depending on the combined biochar (B)-nitrogen (N) fertilization. Within 1 year of treatment, biochar combinations in the soil increased the content of organic carbon; however, on the other hand, they reduced the extraction of humic substances in the SOM. Ultimately, the B-N combinations did not affect the size fractions of soil aggregates after the first year of its incorporation into the soil. The opposite situation was found after nine years, when the SOM, the humic substances (quality and stability), and the content of individual size fractions of macroaggregates showed a balanced response across all treatments. After nine years, a positive trend of decreasing microaggregates content was observed following the application of a higher rate of biochar, as well as both rates of biochar and both levels of N fertilization, which resulted from an increase in the content of humic substances in the soil. Significant changes were observed in the relationships between SOM, humic substances, and aggregate size fractions. Gradual strengthening or changes in the intensity of positive or negative relationships between them were registered, or even a reversal of mutual relationships was recorded due to the aging of biochar application and its combination in the soil environment. The observed changes suggest that biochar demonstrates time-dependent interactions with soil organic matter and structure, as evidenced by PCA and correlation shifts, modifying its effects rather than having a uniform, linear impact. The content of organic carbon due to biochar application and its treatments did not have any positive significant effect on changes in the individual size fractions of aggregates. Changes in the contents of soil aggregates were significantly influenced primarily by the quality of humic substances. Structurally, more condensed and aromatic humic substances from SOM or as part of humus had a positive effect on soil aggregates on one hand, while fulvic acids had the opposite effect. These findings from a field study highlight the potential of biochar as a long-term soil amendment to balance and improve the dynamics of SOM, humic substances, and soil structure. Limitations and Future Scope : Further research is needed to assess its long-term stability and interactions with other soil factors under various environmental conditions. Emphasis should be placed on the stability and water resistance of soil structure and a better understanding of the mechanism of forming stable and water-resistant aggregates in the soil after the application of different rates of biochar and its combinations with different levels of N fertilization as a perspective that can contribute to sustainable soil management and increased agricultural productivity. Declarations Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: All authors consent to the publication of this manuscript. Competing interests: The authors declare no competing interests. Funding: This research was partially supported by the Slovak Research and Development Agency under the contract No. APVV-21–00890, and the Slovak Grant Agency (VEGA) project no. 1/0116/21. Authors' contributions: Vladimír Šimanský : Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review and editing, Supervision. Elżbieta Wójcik-Gront: Statistical analysis, Data curation, Validation, Visualization, Writing - original draft. Sanandam Bordoloi: Data curation, Validation, Writing - review and editing. Ján Horák: Data curation, Validation, Writing - review and editing, Resources, Project administration, Funding acquisition. Acknowledgments: The authors thank the Slovak University of Agriculture for providing the experimental site and technical support. The authors also would like to very much thank the editor and the reviewers for constructive comments. 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Igaz D, Šimanský V, Horák J, Kondrlová E, Domanová J, Rodný M, Buchkina N (2018) Can a single dose of biochar affect selected soil physical and chemical characteristics? J Hydrol Hydromech 66: 421–428. Ippolito J A, Spokas K A, Novak J M, Lentz R D, Cantrell K B (2015) Biochar elemental composition and factors influencing nutrient retention. In: Lehmann J, Joseph S (Eds.) Biochar for Environmental Managament, pp. 139–163. Routledge, Taylor and Francis Group, London, New Yourk. Itami K, Kyuma K (1995) Dispersion behaviour of soils from reclaimed lands with poor soil physical properties and their characteristics with special reference to clay dispersion. Soil Sci Plant Nutr 41: 45–54. Jien S H, Wang Ch S (2013) Effects of biochar on soil properties and erosial potencial in a higly weathered soil. Catena 110: 225–233. Jones D L, Rousk J, Edwards-Jones G, De Luca T H, Murphy D V (2012) Biochar mediated chan ges in soil quality and plant growth in a three year field trial. Soil Biol Biochem 45: 113–124. Juriga M, Šimanský V (2018) Effect of biochar on soil structure – review. Acta Fytotechn Zootechn 21: 11–19. Juriga M, Šimanský V, Horák J, Kondrlová E, Igaz D, Polláková N, Buchkina N, Balashov E (2019) The effect of different rates of biochar and biochar in combination with N fertilizer on the parameters of soil organic matter and soil structure. J Ecol Engin 19: 153–161. Kalu S, Seppänen A, Mganga K Z, Sietiö O M, Karhu K (2024) Biochar reduced the mineralization of native and added soil organic carbon: evidence of negative priming and enhanced microbial carbon use efficiency. Biochar 6: 7. Kan Z R, Liu W X, Liu W S, Lal R, Dang Y P, Zhao X, Zhang H L (2022) Mechanisms of soil organic carbon stability and its response to no-till: A global synthesis and perspective. Glob Chang Biol 28: 693–710. Lal R (2004) Soil carbon sequestration to mitigate climate change. Geoderma 123: 1–22. Lal R, Shukla M K (2004) Principles of soil physics. Marcel Dekker, New York. Lehmann J, Kleber M (2015) The continuous nature of soil organic matter. Nature 528: 60–68. Letey J (1991) The study of soil structure—Science or art. Aust J Soil Res 29: 699 – 707. Li B, Guo Y, Liang F, Liu W, Wang Y, Cao W, Song S, Chen J, Guo J (2024) Global integrative meta-analysis of the responses in soil organic carbon stock to biochar amendment. J Environ Manag 351: 119745. Li H, Yutong W, Tianpei W, Hongrui M (2015) Effect of biochar on organic matter conservation and metabolic quotient of soil. Environ Progr Sustain Energy 34: 1467–1472. Li S, Yang F, Li J, Cheng K (2020) Porous biochar-nanoscale zero-valent iron composites: synthesis, characterization and application for lead ion removal. Sci Total Environ 746: 141037. Mamedov A I, Fujimaki H, Tsunekawa A, Tsubo M, Levy G J (2021) Structure stability of acidic Luvisols: Effects of tillage type and exogenous additives. Soil Till Res 206: 104832. Meng X, Zheng E, Hou D, Qin M, Meng F, Chen P, Qi Z (2024) The effect of biochar types on carbon cycles in farmland soils: A meta analysis. Sci Total Environ 930: 172623. Mierzwa-Hersztek M, Gondek K, Kopieć M, Ukalska-Jaruga A (2018) Biochar changes in soil based on quantitative and qualitative humus compounds parameters. Soil Sci Ann 69: 234–242. Mu D, Mu L, Geng X, Mohamed TA, Wei Z (2024) Evolution from basic to advanced structure of fulvic acid and humic acid prepared by food waste. Int J Biol Macromol 256: 128413. Nguyen B T, Lehmann J, Hockaday W C, Jo Seph S, Masiello C A (2010) Temperature sensitivity of black carbon decomposition and oxidation. Environ Sci Technol 44: 3324–3331. Oades J M (1984) Soil organic mater and structural stability: Mechanisms and implications for management. Plant Soil 76: 319–337. Onweremadu E U, Onyia V N, Anikwe M AN (2007) Carbon and nitrogen distribution in water-stable aggregates under two tillage techniques in Fluvisols of Owerriarea, southeastern Nigeria. Soil Till Res 97: 195–206. Polláková N, Šimanský V, Kravka M (2018) The influence of soil organic matter fractions on aggregates stabilization in agricultural and forest soils of selected Slovak and Czech hilly lands. J Soils Sediments 18: 2790–2800. Rodrigues C I D, Brito L M, Nunes L JR (2023) Soil carbon sequestration in the context of climate change mitigation: A eeview. Soil Systems 7: 64. Sharma P (2024) Biochar application for sustainable soil erosion control: a review of current research and future perspectives. Front Environ Sci 12: 1373287. Shen Z (2024) An overview of biochar application in soil to immobilize heavy metals. In: Shen Z (Ed.) An Overview of Biochar Application in Soil to Immobilize Heavy Metals. Fundamental and Casde Studies. pp. 1–7. Elsevier, Amsterdam, Netherlands. Siebers N, Voggenreiter E, Joshi P, Rethemeyer J, Wang L (2024) Synergistic relationships between the age of soil organic matter, Fe speciation, and aggregate stability in an arable Luvisol. J Plant Nutr Soil Sci 187: 77–88. Stevenson J F (1994) Humus chemistry. John Wiley & Sons, New York, NY, USA. Šimanský V ( 2014) Short communication to the determination of soil structure. Acta Fytotechn Zootechn 17: 1–5. Šimanský V, Juriga M, Golian M, Šlosar M, Provazník M (2021) Soil structure as a significant indirect factor affecting crop yields. Acta Fytotechn Zootechn 24: 129–136. Šimanský V (2016) Effects of biochar and biochar with nitrogen on soil organic matter and soil structure in Haplic Luvisol. Acta Fytotechn Zootechn 19: 129–138. Šimanský V, Polláková N, Chlpík J, Kolenčík M (2023a) Soil science. SPU, Nitra, Slovakia. (in Slovak). Šimanský V, Šrank D, Jonczak J, Juriga M (2019) Fertilization and application of different biochar types and their mutual interactions influencing changes of soil characteristics in soils of different textures. J Ecol Engin 20: 149–164. Šimanský V, Wójcik-Gront E, Rustowska B, Juriga M, Chlpík J, Macák M (2023) Reducing machine movement intensity in the field improves soil structure. Acta Fytotechn Zootechn 26: 93–101. Šrank D, Šimanský V (2020) Differences in soil organic matter and humus of sandy soil after application of biochar substrates and combination of biochar substrates with mineral fertilisers. Acta Fytotechn Zootechn 23: 117–124. Váchalová R, Kolář L, Muchová Z (2016) Primary Soil Organic Matter and Humus, Two Components of Soil Organic Matter. SUA, Nitra, Slovakia, (in Czech). Weber J (2020) Humic substances and their role in the environment. EC Agric 1: 3–8. Weil R R, Brady NC (2017) The nature and properties of soils. Pearson Education Limited, London, UK. Xu, G., Sun, J., Shao, H., Chang, S.X., 2014. Biochar had effects on phosphorus sorption and desorption in three soils with differing acidity. Ecol. Eng. 62, 54–60. Zhang, J., Lü, F., Shao, L., He, P., 2014. The use of biochar-amended composting to improve the humification and degradation of sewage sludge. Bioresour. Technol. 168, 252–258. Zhang, J., Zhang, S., Niu, C., Jiang, J., and Sun, H., 2022. Positive effects of biochar on the degraded forest soil and tree growth in China: a systematic review. Phyton 91(8), 1601–1616. Zhao, S., Ta, N., Li, Z., Yang, Y., Zhang, X., Liu, D., Zhang, A., Wang, X., 2017. Varying pyrolysis tem perature impacts application eff ects of biochar on soil labile organic carbon and humic substances. Appl. Soil Ecol. 116, 399–409. Tables Table 2 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7091756","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485540598,"identity":"f8974637-293f-4be8-b78f-fe0ea1495cad","order_by":0,"name":"Vladimír Šimanský","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHklEQVRIie2RMUvEMBTH31Folx5Z00HrR3hSqCfch2kXpwMHwekMhePicp5fRZfiWAm0S90butwtTroe3qCYUpUDE7hRJL8lLyG/l/8jABbLX8QDoAlQVQ2ybo/gqTXpSt+gOL8Uv9hD2d1j16HHoKBw1s16MjoHIuZ32wcWoXyJV6vpFYSLwqC40SjN6WlGUy5vahFjOznBpKwAnzKtEswgpkpReQa8GfJirJSYJm4JSPTBgpm36RXyyOU7Z2OUtVI+Sghv9Qpx/K9XIOXtkDsxNt0JnwIYginlopsFXXWtPeAiCuqzS5ouCx9r/fiud30vtzlDQqpn+crZ8bISefC2YYfhItEn+3G/i6O+tzB9pIawn4Dtb1gsFst/5xPIWVx7+jSj1QAAAABJRU5ErkJggg==","orcid":"","institution":"Slovak University of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Vladimír","middleName":"","lastName":"Šimanský","suffix":""},{"id":485540599,"identity":"cf5afb7b-b245-463b-8a81-2fd24a1dfb30","order_by":1,"name":"Elżbieta Wójcik-Gront","email":"","orcid":"","institution":"Warsaw University of Life Sciences— 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2","display":"","copyAsset":false,"role":"figure","size":762133,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7091756/v1/334705df82af130613004183.png"},{"id":86932360,"identity":"7d80278c-3e54-452a-89f5-8d10d7cbff1c","added_by":"auto","created_at":"2025-07-17 10:00:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":384429,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7091756/v1/f1c893b9d9c029fe693f1ada.png"},{"id":86932361,"identity":"2561a746-0e4c-4df1-9d0b-dc610c856afa","added_by":"auto","created_at":"2025-07-17 10:00:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264716,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7091756/v1/bc75cfd16743a6605ee8109b.png"},{"id":95039771,"identity":"565f4153-e25d-4cfa-b384-dfb9c67bc2d9","added_by":"auto","created_at":"2025-11-03 16:00:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2599013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7091756/v1/b61386a9-527a-4069-b7ea-61d169b872be.pdf"},{"id":86932363,"identity":"b040203e-2c67-4c20-be9c-3605d6912a90","added_by":"auto","created_at":"2025-07-17 10:00:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32620,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7091756/v1/e5476979ee5d91049e9d0a58.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biochar and its combination with nitrogen fertilization altered soil organic matter, humic substances, and soil structure: Short-term vs. long-term changes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMultiple perspectives and approaches have existed regarding soil structure and humic substances in agricultural soils, with some being more or less preferred by soil scientists. For example, Amezketa (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and Bronick and Lal (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), in their reviews on soil structure, addressed a wide range of environmental and anthropogenic factors and their interactions, emphasizing soil aggregates and their stability. Numerous recent scientific studies on soil structure (aggregate stability and its measurement) follow this direction (Šimansk\u0026yacute;, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Memedov et al., 2021; Šimansk\u0026yacute; et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Siebers et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, Levy (1991) presented a different perspective on soil structure, focusing on the arrangement of soil pores and highlighting the importance of their unique architecture with time. Regardless, soil structure is a fundamental physical property that deserves attention (Foth, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Lal and Shukla, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Blume et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, the study of humus in soil, despite being a subject of scientific inquiry for over 200 years, has seen various perspectives and approaches (Stevenson, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Lehmann and Kleber, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The most crucial aspect is that humus is a component of soil organic matter (Stevenson, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Weil and Brady, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), playing critical roles in soil formation and its physical, nutritional, chemical, and biological properties (Pol\u0026aacute;kov\u0026aacute; et al., 2018; Mu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Literature corroborates that soil structure largely results from the content and quality of humic substances in the soil (Pol\u0026aacute;kov\u0026aacute; et al., 2018). An existing knowledge gaps have been identified concerning the relationships between soil structure and humic substances following the application of biochar, biochar substrates, or their combinations with mineral fertilization (Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mierzwa-Hersztek et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Juriga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBiochar generally refers to a type of porous, highly aromatic, insoluble substance produced by the pyrolysis of biomass, such as lignocellulosic biomass and plant waste, under low-oxygen or anoxic conditions (Hansen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Biochar has a high content of stable carbon, which resists decomposition and remains in the soil for a long time, contributing to carbon sequestration (Hossain et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Šrank and Šimansk\u0026yacute;, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to its high specific surface area, micropore content (Chintala et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), lower bulk density (Glab et al., 2016), and the presence of functional groups such as phenolic hydroxyl groups, alcoholic hydroxyl groups, and carbonyl groups, etc. (Xu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Glab et al., 2016), as well as other benefits (regulation and immobilization of harmful substances such as heavy metals \u0026ndash; Shen, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), biochar can be applied to soil as an additive that manages water regime (Igaz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sharma, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), nutrient regime (Chen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), cation exchange capacity and soil sorption properties (Igaz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hossain et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and reduces soil bulk density (Glab et al., 2016). From soil physical properties, biochar applied to soil can influence soil structure (Juriga and Šimansk\u0026yacute;, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) by improving soil aggregation and creating a more stable and porous soil matrix (Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Biochar also enhances soil structure by reducing soil compaction, especially in texturally heavy soils with higher clay content (Blanco-Canqui, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The resulting favorable soil structure provides an excellent foundation for root growth and microbial activity, potentially benefiting crop performance through higher crop yields.\u003c/p\u003e\u003cp\u003eBiochar, as well as its combination with nitrogen (N) fertilization, can enhance soil properties, thereby improving the quality and fertility of arable land. We have hypothesized three scenarios on how it may enhance that. First, since humus formation is a long-term process, the effect of biochar and its combination with N fertilization on humus formation will be more pronounced after prolonged residence in the soil (H1). Second, assuming that the prolonged presence of biochar in the soil would improve soil structure by reducing microaggregates and increasing mainly favorable soil macroaggregates in a size fraction of 0.5-3 mm over time (H2). Third, changes in soil organic matter and humic substances may have a direct impact on the size fractions of soil aggregates (H3). Based on the context and hypotheses, the objectives of this study were (i) to determine the extent to which biochar and its combination with N fertilization on the content and quality of humic substances and soil structure and (ii) to quantify changes in the relationships between humic substances and soil structure.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Site description\u003c/h2\u003e\u003cp\u003eThe Slovak University of Agriculture's experimental site in Nitra (Doln\u0026aacute; Malanta) is located at 48\u0026deg;10'00\" N, 18\u0026deg;09'00\" E, in the northeastern Danubian Lowland and the western Žitava Upland, near the lower basin of the Selenec stream. This site is 4 km east of Nitra, within a warm maize-growing region. The experimental area features flat terrain with a gentle southwest slope at an elevation of 170\u0026ndash;180 meters above sea level. Geologically, it sits at the boundary between the crystalline-Mesozoic massif of the Tribeč Mountains and the Žitava Upland. The geological base is primarily composed of eluvial-deluvial sediments from the Tribeč Mountains, mixed with loess sediments from the Žitava Upland in some areas. The soil type is Haplic Luvisol, with a silt loam texture in the A-horizon. The soil contains an average of 360.4 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sand, 488.3 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e silt, and 151.3 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e clay and has a slightly acidic pH of 5.71. Before the experiment began, the soil had low organic carbon content (9.13 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), a moderately low cation exchange capacity (142 mmol p\u003csup\u003e+\u003c/sup\u003e kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and a saturated sorption complex (85%). The site is in a very warm and dry agro-climatic region, with an average annual precipitation of 559 mm and an average annual temperature of 10.7\u0026deg;C (based on data from 1991\u0026ndash;2020).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental design\u003c/h2\u003e\u003cp\u003eThe biochar experiment was established in 2014, prior to the sowing of spring barley, and is ongoing. The crop rotation was as follows: 2014 - spring barley, 2015 - maize, 2016 - spring wheat, 2017 - maize, 2018 - spring barley, 2019 - maize, 2020 - peas, 2021 - winter wheat, 2022 - maize, and in 2023 - spring barley. The original experiment consists of 9 treatments detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The experiment was set up using a randomized block design with three replications for each treatment. Each plot measured 6 x 4 m and was separated by a 1 m protective strip. Biochar was applied to the soil surface in the spring of 2014 at the respective treatment rate, manually spread over each plot, and then incorporated into the soil to a depth of 10 cm using a combinator. Biochar was applied at rates of 10 and 20 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (B10 and B20). The first level of nitrogen fertilization (N1) was determined based on the requirements of the specific crop grown in that year and calculated as per practices in the Slovak Republic. The second level of nitrogen fertilization (N2) was increased by 100%. Throughout the experiment, nitrogen fertilization rates ranged from 30 to 160 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for N1 and from 45 to 240 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for N2. Specifically, in 2015, the rates were 160 and 240 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for N1 and N2, respectively, and in 2023, the rates were 40 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for N1 and 80 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for N2. Nitrogen fertilization was always carried out during the growing season of the cultivated crop, with the nitrogen doses divided into 2\u0026ndash;3 applications depending on the amount of nitrogen. The soil was not plowed during the experiment; it was only disked or surface-tilled to a maximum depth of 15 cm. Standard agronomic practices during the growing season of the cultivated crops were carried out each year (including disease, pest control, and weeding).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental design for the current study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSl. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDesignation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB0N0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eno biochar, no nitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB10N0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 10 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and no nitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB20N0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 20 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and no nitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB10N1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 10 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a first level of N fertilization\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB20N1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 20 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a first level of N fertilization\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB10N2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 10 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a second level of N fertilization\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB20N2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebiochar at a rate of 20 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a second level of N fertilization\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Biochar and nitrogen mineral fertilizers\u003c/h2\u003e\u003cp\u003eThe applied biochar was produced from cereal husks and waste sludge generated during paper production. The pyrolysis temperature was 550\u0026deg;C with a duration of 30 minutes. The biochar contained 53.1% total carbon, 1.4% total nitrogen, 57 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e calcium, 3.9 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e magnesium, 15 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e potassium, 0.7 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sodium, and 38.3% ash. The specific surface area was 21.7 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Its pH was 8.8, and the particle size of the produced biochar ranged from 1 to 5 mm. Nitrogen was applied in the form of the following commercial mineral fertilizers: LAD27, DASA 26/13, and NPK.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Soil sampling and analysis\u003c/h2\u003e\u003cp\u003eFor the purposes of sampling in this study, soil samples were collected from a depth of 20 cm from each replication of the respective treatment at monthly intervals from April to September in 2015 and from April to July in 2023. Soil samples were taken to determine the size fractions of soil aggregates and parameters of soil organic matter (SOM). The soil samples were carefully collected using a spade to minimize damage to the soil aggregates. After drying, the samples were sieved through a set of sieves 3, 0.5, and 0.25 mm. The aggregates remaining on the sieve were then weighed and expressed as a percentage of the total soil weight. The following size fractions of dry soil aggregates (DSA) were obtained: \u0026gt;3 mm, 3-0.5 mm, 0.5\u0026thinsp;\u0026minus;\u0026thinsp;0.25 mm, and \u0026lt;\u0026thinsp;0.25 mm. The size threshold separating macroaggregates (ma) from microaggregates (mi) is 0.25 mm. Agronomically valuable macroaggregates are in the 0.5-3 mm range (Fulajt\u0026aacute;r, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Šimansk\u0026yacute; et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea). Soil samples for determining SOM and humus were homogenized, ground, sieved through a 0.25 mm sieve, and then analyzed using standard methods. Soil organic carbon (Corg) was quantified using the wet combustion technique, which involves the oxidation of soil organic matter (SOM) with a mixture of 0.07 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, followed by titration with Mohr\u0026rsquo;s salt (Dzadowiec and Gonet, 1999a). The group and fraction composition of humic substances (HSs) including humic acids (HAs) and fulvic acids (FAs) were analyzed using the method described by Belchikova and Kononova, which includes extraction with a mixture of 0.01 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Na\u003csub\u003e4\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, 10 H\u003csub\u003e2\u003c/sub\u003eO, and 0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH (Dzadowiec and Gonet, 1999b). The light absorbance of HSs and HAs at wavelengths of 465 and 650 nm was measured using a Jenway 6400 Spectrophotometer to determine the color quotients of humic substances (Q\u003csup\u003e4/6\u003c/sup\u003e\u003csub\u003eHS\u003c/sub\u003e) and humic acids (Q\u003csup\u003e4/6\u003c/sup\u003e\u003csub\u003eHA\u003c/sub\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e\u003cp\u003eFirst, a two-way ANOVA was applied to determine whether year and treatment significantly influenced the observed variables. Subsequently, sampling time (April-September for 2015 and April-July for 2023) was treated as a repetition in the one-way ANOVA conducted separately for each year, with treatment as the factor. Analysing each year separately avoids the assumption that treatments had identical effects in both years, allowing for a more nuanced understanding of treatment impacts. Pearson\u0026rsquo;s correlation analysis was performed to examine relationships between different measured variables within each treatment for 2015 and 2023 separately. Principal Component Analysis (PCA) was conducted to reduce the dimensionality of the dataset while preserving the most significant variance. The first two principal components (PC1 and PC2) were selected for visualization and interpretation, as they accounted for the largest proportion of variance. A biplot was used to illustrate the relationships between treatments and variables, highlighting major patterns in the dataset and demonstrating how experimental treatments affected variable relationships and overall variance.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effect of biochar on soil organic matter, humic substances, and soil structure\u003c/h2\u003e\u003cp\u003eThe Corg content was generally higher in 2023 than in 2015 (Fig.\u0026nbsp;1), indicating a moderate improvement in soil management practices and aligning with current global challenges such as sustainable agriculture, carbon neutrality (Lal, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Rodrigues et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The Corg content in B0N0 (control treatment) increased from 1.22% in 2015 to 1.51% in 2023. Soil management alternates Corg in soils (Rodrigues et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), leading to various scenarios ranging from negative through neutral and stable to positive (Weil and Brady, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Overall, the positive scenario in this study was attributed to several factors, such as no-tillage and reduced tillage intensity (Kan et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leaving crop residues on the soil surface (Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and the application of biochar (Šrank and Šimansk\u0026yacute;, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The effects of biochar application and its combination with N fertilization on changes in Corg content differed between 2015 and 2023. In 2015, Corg content increased by 32%, 21%, 51%, 31%, and 34% in B20N0, B10N1, B20N1, B10N2, and B20N2, respectively, compared to B0N0. It appears that biochar application increases soil C as a result of the negative priming effect of SOM by biochar and its combination with N fertilization (Kalu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biochar is a source of stable C (Meng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and its incorporation into the soil can reduce SOM mineralization. However, labile C, formed during SOM decomposition by microbial activity, can be sorbed onto biochar (Jones et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), thus increasing Corg content as documented by the results of this study (Fig.\u0026nbsp;1). In this study, a higher rate of biochar was more effective than the lower rate in increasing Corg. Adequate nitrogen is essential for biological processes and supporting microbial activity. Biochar can contain nutrients, including nitrogen (Ippolito et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which can influence the mineralization of SOM and biochar particles in the soil. Corg content was relatively balanced among treatments in 2023, suggesting that properly set soil management was more decisive than the persistent effect of biochar and its combination with N fertilization. The suppressed effect of biochar on Corg content could be due to its stability and reduced surface activity, as biochar particles were part of soil aggregates (Šimansk\u0026yacute;, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and soil casts after increased earthworm activity (Šimansk\u0026yacute; et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In 2015, the extraction of humic substances in SOM (extHS) decreased with the subsequent increase in Corg, indicating reduced mineralization and humification of SOM and the subsequent accumulation of Corg in the soil due to the negative priming effect (Kalu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The extraction of humic acids in SOM (extHA) as part of HSs significantly decreased only in B20N1, B10N2, and B20N2 treatments. The extraction of fulvic acids in SOM (extFA) significantly decreased only because of the application of a higher rate of biochar and its combination with the first level of N fertilization in 2015. In 2023, extHS, including extHA and extFA, did not change depending on the treatment (Fig.\u0026nbsp;1). These results indicate that while biochar increased the content of Corg in the soil, it did not affect the extraction of HSs in SOM, confirming that biochar is characterized by lower reactivity and a more stable structure (Nguyen et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gupta and Germida, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The degree of humification of organic matter after the application of biochar and its combination with N ranged from 15\u0026ndash;24% and from 20\u0026ndash;24%, corresponding to a low to medium degree of humification in 2015 and 2023, respectively. It is evident that primary organic matter predominates over humic substances in the soil under treatments, which generally mineralizes more and humifies significantly less (V\u0026aacute;chalov\u0026aacute; et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Based on the obtained results, it is evident that the application of biochar and its combination with N fertilization did not clearly enhance the formation of humic substances in SOM; rather, it reduced humification. H1 was partially supported because, in 2015, a reduction in humification was observed under all treatments with biochar and its combinations with nitrogen fertilization. After 9 years, there was a trend of increasing extraction of HS and HA in SOM under treatments B10N0, B10N1, B10N2, and B20N2.\u003c/p\u003e\u003cp\u003eThe utilization of various types of biochar has great potential for the formation and stabilization of humic substances (Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mierzwa-Hersztek et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which was partially supported in this study (Fig.\u0026nbsp;2). Through biochar and its combination with N fertilization, humic substances were not added to the soil. No significant differences were observed between 2015 and 2023, i.e., 1 and 9 years after biochar application. A moderate increase in C\u003csub\u003eHS\u003c/sub\u003e was found only in 2023 after the application of 20 t of biochar ha⁻\u0026sup1; and an increased level of N fertilization (N2). In other treatments, an insignificant trend in the increase of C\u003csub\u003eHS\u003c/sub\u003e was observed. The C\u003csub\u003eHA\u003c/sub\u003e:C\u003csub\u003eFA\u003c/sub\u003e ratio in all treatments, including the control, was above 1, indicating overall favorable humus quality in the soil at the experimental field. The C\u003csub\u003eHA\u003c/sub\u003e:C\u003csub\u003eHA\u003c/sub\u003e ratio did not change significantly in any year. Nevertheless, an insignificant trend of improving the C\u003csub\u003eHA\u003c/sub\u003e:C\u003csub\u003eFA\u003c/sub\u003e ratio in favor of C\u003csub\u003eHA\u003c/sub\u003e was observed after the application of biochar, while a higher level of N fertilization tended to decrease the C\u003csub\u003eHA\u003c/sub\u003e:C\u003csub\u003eFA\u003c/sub\u003e ratio. Humus in the soil is relatively stable (Stevenson, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Weil and Brady, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but due to external influences, such as the application of biochar in this study, its stability can be disrupted, and soil humus begins to be attacked by soil microorganisms (Adani et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), resulting in a reduction of humic substances in the soil (Mierzwa-Hersztek et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Of course, the properties of biochar have a crucial impact on these processes (Zhao et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and is also dependent on the biochar type. In all treatments, the color quotient values of humic substances and humic acids suggested more humified and mature organic matter in the soil, with a high content of condensed aromatic compounds and a low representation of aliphatic compounds (Weil and Brady, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Weber, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The molecular weight and degree of condensation of humic substances remained unchanged (Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eSoil structure is fundamentally modified by external factors in addition to internal factors (Am\u0026eacute;zketa, 1999; Bronick and Lal, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Among external influences within agronomic practices, the application of farmyard manure, composts (Šimansk\u0026yacute; et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), or organic amendments such as biochar (Juriga and Šimansk\u0026yacute;, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Baiamonte et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) is recommended to improve soil structure. Reactive surface functional groups of biochar can form organo-mineral complexes with finer soil particles (clay), which are the basis of soil microaggregates (Brodowski et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Soil structure formation is mostly associated with microbial activity. Another mechanism is explained through microscopic fungi that can colonize and grow in the pores of biochar, thus supporting the formation and stability of soil structure (Cross et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The aggregation process is also significantly influenced by the properties of the applied biochar. N fertilization applied with biochar can act as an accelerator, speeding up the mineralization of SOM and biochar particles, improving microbial activity, and resulting in soil aggregate formation (Šimansk\u0026yacute;, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, soil structure formation in the case of biochar is also the result of external influences, including climatic conditions. In this study, the application of biochar in both rates and its combination with both levels of N fertilization (N1, N2) did not have a statistically significant effect on changes in DSAma content 1 and 9 years after their application. DSAma contents\u0026thinsp;\u0026gt;\u0026thinsp;3 mm were significantly higher in 2023 than in 2015 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas DSAma contents 0.25-3 mm were significantly higher in 2015 than in 2023 across all treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The results suggest that climatic conditions had a greater impact on soil structure than biochar itself (Fig.\u0026nbsp;3). Climate, through cycles of soil drying and wetting, as well as freezing and thawing cycles, has a significant effect on soil structure (Foth, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Lal and Shukla, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Bronick and Lal, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Blume et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In terms of total precipitation and average temperatures, the years 2015 and 2023 were evaluated as moisture-normal, extremely warm and extremely wet, and very warm, respectively, compared to the climatic norm of 1991\u0026ndash;2020 (unpublished data). Temperature and moisture changes reflect the activity of soil microorganisms, which have a crucial impact on aggregation and structure formation (Bronick and Lal, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). DSAmi contents were significantly lower in 2023 than in 2015 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, in 2015, biochar and its combinations with N fertilization had no effect on DSAmi, whereas in 2023, i.e., 9 years after its incorporation into the soil, a positive effect was identified. DSAmi contents after the application of 20 t of biochar ha⁻\u0026sup1; decreased by 33% and 30% compared to B0N0 (control) and B10N0, respectively. This suggested that microaggregates bonded with biochar particles into larger macroaggregate size fractions, which were evenly balanced across treatments (Fig.\u0026nbsp;3). Overall, the effects on soil structure are varied and tend to manifest after long-term biochar application in the soil rather than in a short time interval after its incorporation (Jien and Wang, 2013), as documented by the findings of this study (Fig.\u0026nbsp;3). Based on the results of this study, H2 was partially supported because, after the first year, no trends or significant changes in soil aggregate contents were observed under the individual treatments. After 9 year from the establishment of the experiment, it was found that the content of microaggregates was significantly reduced in the B20N2 treatment, and a trend of decreasing microaggregate content was observed in treatments such as B20N0, B10N1, B20N1, and B10N2. However, this did not translate into a substantial increase in agronomically valuable size fractions of macroaggregates (0.5-3 mm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Relationships between soil organic matter, humic substances, and soil structure\u003c/h2\u003e\u003cp\u003eSOM and aggregation are closely related, as SOM influences soil structure stability by acting as a binding agent that connects mineral particles, reduces aggregate wettability, and thus affects their mechanical strength (Onweremadu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Soil organic carbon binds with clay particles through polyvalent cations, forming an organo-mineral complex, which is the foundation of soil structure (Bronick and Lal, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Humic substances within SOM have a significant effect on soil structure (Poll\u0026aacute;kov\u0026aacute; et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Weber, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as does the application of biochar and its combination with N fertilization (Juriga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As previously mentioned, biochar is a source of stable carbon with lower reactivity and a more stable structure (Nguyen et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gupta and Germida, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Meng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When biochar is applied with readily available N, it can promote SOM mineralization through microorganisms (Ali et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which produce binding agents responsible for connecting soil particles, resulting in improved soil structure (Costa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, the relationships between SOM, humic substances, and aggregate size fractions varied across all treatments depending on the year (Table\u0026nbsp;2), potentially due to changes in fundamental biological mechanisms. Notably, no statistically significant correlations were found between Corg and individual size fractions of DSAma and DSAmi, either 1 or 9 years after biochar application. This phenomenon is likely related to the quality of SOM. Oades (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) stated that not all organic compounds in soil are responsible for aggregation. Different forms of organic matter stabilize aggregates of various sizes and sometimes may not influence soil aggregation at all. For example, anions of organic compounds such as fulvates, citrates, oxalates, or acetates increase clay dispersion, while aromatic acids (salicylic acid, p-hydroxybenzoic acid) have a flocculating effect (Itami and Kyuma, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). This is partially supported by the findings of this study, where primarily lower contents of extFA in SOM and CFA, because of the effect of biochar applied at a rate of 10 t ha⁻\u0026sup1; and its combination with both levels of N fertilization after 1 year (Fig.\u0026nbsp;1), increased the contents of individual size fractions of DSAma and DSAmi (Table\u0026nbsp;2). The relationships between SOM, humic substances, and soil structure were markedly different depending on the treatment. For example, B10N0 was associated with high values of variables moving in the same direction (DSAma 0.5\u0026thinsp;\u0026minus;\u0026thinsp;0.25 mm in 2015, CHA in 2015). This treatment was significantly different from B20N2, where the opposite trends were observed (Fig.\u0026nbsp;4). Across all treatments, the overall number of significant relationships was higher in 2015 than in 2023 and often differed markedly within treatments (Table\u0026nbsp;2; Fig.\u0026nbsp;4). This suggests complex interactions between internal factors (SOM, humic substances, soil structure) and external factors (weather conditions affecting soil temperature and moisture). H3 was partially supported because a significant relationship between the monitored parameters of SOM, humic substances, and size fractions of soil aggregates was not consistently observed across treatments and years.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe results indicate that the parameters of soil organic matter (SOM), humic substances, and soil structure, as well as their long-term interrelationships, significantly varied depending on the combined biochar (B)-nitrogen (N) fertilization. Within 1 year of treatment, biochar combinations in the soil increased the content of organic carbon; however, on the other hand, they reduced the extraction of humic substances in the SOM. Ultimately, the B-N combinations did not affect the size fractions of soil aggregates after the first year of its incorporation into the soil. The opposite situation was found after nine years, when the SOM, the humic substances (quality and stability), and the content of individual size fractions of macroaggregates showed a balanced response across all treatments. After nine years, a positive trend of decreasing microaggregates content was observed following the application of a higher rate of biochar, as well as both rates of biochar and both levels of N fertilization, which resulted from an increase in the content of humic substances in the soil.\u003c/p\u003e\u003cp\u003eSignificant changes were observed in the relationships between SOM, humic substances, and aggregate size fractions. Gradual strengthening or changes in the intensity of positive or negative relationships between them were registered, or even a reversal of mutual relationships was recorded due to the aging of biochar application and its combination in the soil environment. The observed changes suggest that biochar demonstrates time-dependent interactions with soil organic matter and structure, as evidenced by PCA and correlation shifts, modifying its effects rather than having a uniform, linear impact. The content of organic carbon due to biochar application and its treatments did not have any positive significant effect on changes in the individual size fractions of aggregates. Changes in the contents of soil aggregates were significantly influenced primarily by the quality of humic substances. Structurally, more condensed and aromatic humic substances from SOM or as part of humus had a positive effect on soil aggregates on one hand, while fulvic acids had the opposite effect. These findings from a field study highlight the potential of biochar as a long-term soil amendment to balance and improve the dynamics of SOM, humic substances, and soil structure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations and Future Scope\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eFurther research is needed to assess its long-term stability and interactions with other soil factors under various environmental conditions. Emphasis should be placed on the stability and water resistance of soil structure and a better understanding of the mechanism of forming stable and water-resistant aggregates in the soil after the application of different rates of biochar and its combinations with different levels of N fertilization as a perspective that can contribute to sustainable soil management and increased agricultural productivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All authors consent to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was partially supported by the Slovak Research and Development Agency under the contract No. APVV-21\u0026ndash;00890, and the Slovak Grant Agency (VEGA) project no. 1/0116/21.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e \u003cstrong\u003eVladim\u0026iacute;r \u0026Scaron;imansk\u0026yacute;\u003c/strong\u003e: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review and editing, Supervision. \u003cstrong\u003eElżbieta W\u0026oacute;jcik-Gront:\u0026nbsp;\u003c/strong\u003eStatistical analysis, Data curation, \u0026nbsp;Validation, Visualization, Writing - original draft. \u003cstrong\u003eSanandam Bordoloi:\u0026nbsp;\u003c/strong\u003eData curation, \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eValidation,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWriting - review and editing. \u003cstrong\u003eJ\u0026aacute;n Hor\u0026aacute;k:\u003c/strong\u003e Data curation, \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eValidation,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWriting - review and editing, Resources, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors thank the Slovak University of Agriculture for providing the experimental site and technical support. The authors also would like to very much thank the editor and the reviewers for constructive comments.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdani F, Genevini P, Ricca G, Tambone F, Montoneri E (2007) Modyfication of soil humic matter after 4 years of compost application. \u003cem\u003eWaste Manag\u003c/em\u003e 27: 319\u0026ndash;324.\u003c/li\u003e\n\u003cli\u003eAhmad A, Arif M S, Shahzad S M, Yasmeen T, Shakoor A, Iqbal S, Riaz A, Zahid A, Chapman S J (2024) Long-term raw crop residue but not burned residue incorporation improved soil multifunctionality in semi-arid agroecosystems. 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Soil Ecol. 116, 399\u0026ndash;409.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"biochar, fulvic acids, humic substances, soil organic matter, soil aggregates","lastPublishedDoi":"10.21203/rs.3.rs-7091756/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7091756/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiochar (B), as well as its combination with nitrogen (N) fertilization, can influence soil quality and fertility. Humus formation and aggregation is a long-term process in soils and the impact of combined biochar-N fertilization on its formation remains underexplored for long term studies. The aim of this study was to determine the extent to which combined biochar-N fertilization on the soil organic matter (SOM) content, quality of humic substances (HS), and soil structure. We also aimed at quantifying changes in the relationships between HS and soil structure. Silty loam Haplic Luvisol was sampled from the field after 1- and 9- years from the incorporation of biochar (0, 10, and 20 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of biochar marked as B0, B10, B20) combined with N fertilization (N0, N1, and N2). The results showed that B\u0026thinsp;+\u0026thinsp;N fertilization moderately increased the soil organic carbon (Corg) content in the soil after 1 year of incorporation. After 9 years, the Corg content in the soil was relatively balanced among the treatments. Only in B20N2 did the HS content significantly increase compared to B0N0. In B20N2, the content of microaggregates significantly decreased compared to B0N0 after 9 years. Significant changes in correlations between SOM, HS, and aggregate size fractions suggest potential shifts in their relationships over the decade. The gradual strengthening and changes in the intensity of positive or negative relationships between them suggests the aging of biochar may have long term effects on crop productivity and soil health.\u003c/p\u003e","manuscriptTitle":"Biochar and its combination with nitrogen fertilization altered soil organic matter, humic substances, and soil structure: Short-term vs. long-term changes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 10:00:20","doi":"10.21203/rs.3.rs-7091756/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T12:57:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T03:19:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232190282949649725714362779768866360590","date":"2025-07-17T06:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T04:35:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-11T21:21:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-11T18:31:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Geochemistry and Health","date":"2025-07-10T10:01:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"12c1a77b-7f2e-48e3-b5b0-c9838584e2a1","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T15:59:08+00:00","versionOfRecord":{"articleIdentity":"rs-7091756","link":"https://doi.org/10.1007/s10653-025-02853-7","journal":{"identity":"environmental-geochemistry-and-health","isVorOnly":false,"title":"Environmental Geochemistry and Health"},"publishedOn":"2025-10-28 15:56:52","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-07-17 10:00:20","video":"","vorDoi":"10.1007/s10653-025-02853-7","vorDoiUrl":"https://doi.org/10.1007/s10653-025-02853-7","workflowStages":[]},"version":"v1","identity":"rs-7091756","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7091756","identity":"rs-7091756","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}