Human activities threaten to release a significant amount of ecosystem carbon (C) as CO2, impacting the climate. Agriculture plays a major role, releasing substantial amounts of C from soil globally. Plants release a considerable portion of photosynthetically fixed C into the soil, fueling microbial growth and respiration, which produces CO2. Significant soil organic carbon (SOC) loss has been observed in lands converted to tropical grasslands, particularly on Ferralsols. The urgent challenge is to reverse this loss and increase C retention in soil. The Intergovernmental Panel on Climate Change (IPCC) recognizes soil C management and biochar application as potential carbon dioxide removal (CDR) methods, offering benefits like improved soil health and sustained agricultural productivity. Protecting and rebuilding soil C could sequester a substantial amount of CO2 annually. Biochar, a product of biomass pyrolysis, is a persistent CDR method with various environmental benefits. This research assesses biochar's capacity to build new biogenic SOC reserves by examining mechanisms such as the acceleration of organo-mineral and organo-organic interface formation in soil microaggregates and mineral fractions to protect SOC from degradation. The study investigates SOC mineralization, microbial C-use efficiency, spatial distribution of C functional groups, and mineral protection of SOC to quantify biochar's potential to lift the SOC storage ceiling.

The existing literature highlights the soil carbon saturation concept and its implications for SOC storage. Several studies have shown biochar's potential for protecting new soil carbon and improving soil health, however the mechanisms behind the long-term influence of biochar on soil carbon remains unclear. Existing research has explored the roles of mineral protection, organo-mineral associations and the impact of carbon to nitrogen ratios in stabilizing soil organic matter. The interaction between biochar and soil microbes have been explored, including effects on microbial biomass and diversity. There is existing research regarding enzyme activities in soils amended with biochar and the role of root exudates in influencing soil organic carbon dynamics. While previous studies have indicated biochar's positive effects, this research uses advanced microspectroscopic techniques to provide a more detailed mechanistic understanding.

A long-term field experiment (9.5 years) was conducted on a Rhodic Ferralsol under subtropical pasture in Australia. Four treatments were applied: Control (no biochar), Historical (biochar applied once at trial establishment), Control+Recent (biochar applied after 8.2 years to Control plots), and Historical+Recent (biochar applied after 8.2 years to Historical plots). Soil SOC stocks were measured to assess the impact of biochar on SOC storage capacity. Rhizosphere priming (the effect of roots on SOC mineralization) was quantified using a pulse labeling technique with 13C. Aggregate size and density fractionation was performed to partition rhizodeposits, biochar C, and SOC. Microbial biomass, catabolic enzyme activities, and metabolic quotients were measured to assess microbial contributions. Advanced microscopic techniques, including one-dimensional spectroscopic, two-dimensional microspectroscopic, and three-dimensional electron microscopic analyses, were employed to visualize SOC spatial heterogeneity and organo-mineral interactions. Synchrotron-based soft X-ray (SXR) spectroscopy and synchrotron-based infrared microspectroscopy (IRM) were used to characterize C functional groups and their spatial distribution in microaggregates and mineral fractions. Statistical analysis was used to determine significance.

The study found that the SOC storage ceiling could be lifted through single or multiple biochar applications. The Historical plots stored significantly more SOC compared to the Control plots. Adding biochar to the Control plots after 8.2 years increased SOC storage, while a second biochar application further increased SOC. The increase in SOC storage was linked to decreased net cumulative SOC mineralization (negative priming). The Historical+Recent treatment significantly lowered SOC mineralization compared to Control+Recent soils. The analysis of belowground C pools showed increased retention of C in the Historical+Recent treatment, particularly in mineral-protected soil organic matter. Microbial biomass increased in Control+Recent soils, while the Historical+Recent soils exhibited lower metabolic quotients, indicating increased microbial C-use efficiency. Microscopic analyses provided visual evidence of organo-mineral and organic interface formation on biochar surfaces, protecting rhizodeposits and microbial necromass. SXR and IRM analyses revealed spatial heterogeneity of C functional groups, with a higher proportion of aliphatic C in the Historical+Recent treatment. The study estimated that a global application of biochar to Ferralsols under tropical pasture could potentially create a substantial additional soil C sink.

The findings address the research question by demonstrating the capacity of biochar to lift the SOC ceiling. The observed negative priming, increased C retention, and altered microbial C-use efficiency are key mechanisms explaining this effect. The advanced microscopic techniques provided unprecedented insights into the nanoscale and microscale processes driving SOC stabilization. The results have significant implications for global efforts to build SOC and mitigate climate change. The estimated potential for increased CDR through biochar application highlights the potential of this strategy for climate change mitigation. The study's findings provide strong support for the use of biochar as a tool for enhancing SOC storage in (sub)tropical grasslands.

This study demonstrates that strategic biochar application can effectively lift the soil organic carbon ceiling, primarily through mechanisms of negative priming and enhanced organo-mineral complex formation. The findings highlight the potential for large-scale biochar application to significantly enhance soil carbon sequestration and contribute to climate change mitigation. Future research could focus on exploring the broader applicability of these findings across diverse soil types and environments, investigating the long-term stability of biochar-induced SOC increases, and optimizing biochar production and application strategies for maximum impact.

The study was conducted at a single field site, limiting the generalizability of the findings to other geographic locations and soil types. The observed effects might vary depending on factors like biochar type, application rate, climate conditions, and management practices. While the study provides strong evidence for the mechanisms involved, further research is needed to fully understand the complexity of biochar-soil interactions and their long-term effects on SOC dynamics. The extrapolation of the study's findings to the global level relies on certain assumptions that could influence the overall estimations of CDR potential.