Biochar and its combination with nitrogen fertilisation altered soil organic matter, humic substances, and soil structure: short-term versus long-term changes

Biochar (B), particularly when combined with nitrogen (N) fertilisation, can significantly influence soil quality and fertility. However, its long-term effects on soil organic matter (SOM), humic substances (HS), and soil structure remain insufficiently understood. This study examined the impacts of biochar applied at 0, 10, and 20 t ha⁻¹ (B0, B10, B20), in combination with two levels of N fertilization (N1, N2), on a silty loam Haplic Luvisol over short-term (1 year) and long-term (9 years) periods at the Slovak University of Agriculture experimental site (Nitra, Slovakia). After 1 year, biochar treatments increased soil organic carbon (Corg) by up to 51% (B20N1) compared with the control (B0N0), but significantly reduced the extraction of humic substances, particularly in B20N1. After 9 years, Corg contents were relatively balanced across treatments, but B20N2 exhibited a marked increase in HS content and a 33% reduction in microaggregates relative to B0N0, indicating the formation of larger macroaggregates. Principal component and correlation analyses revealed time-dependent changes in the relationships between SOM, HS, and aggregate size fractions. Importantly, almost no consistent correlation was observed between Corg and aggregate size fractions, suggesting that the quality and chemical characteristics of humic substances, especially their aromatic and condensed nature, play a more critical role in soil structure formation than total carbon content alone. This study provides new evidence that biochar's long-term impact on soil structure is mediated not by immediate increases in carbon levels, but by gradual improvements in humus chemistry and aggregate dynamics. Our findings challenge conventional assessments of soil amendments based solely on carbon metrics and highlight the need to consider humic substance quality as a key driver of soil structural resilience.