Effect of vertical depth gradients on soil carbon recalcitrance stratification in a restoring secondary forest.

Soil organic matter (SOM) dynamics are central to evaluating carbon storage and functional recovery in restoring tropical secondary forests. Assessment of carbon form stabilization along vertical soil gradients provides key insights into the progression of essential biogeochemical processes during ecological restoration, such as plant litter decomposition, microbial organic matter processing, and nutrient cycling. This study evaluated the vertical distribution of SOM structural composition in a secondary tropical forest undergoing restoration for 20 years. Soil sampling was conducted beneath the canopy projection of 12 tree individuals across three depth intervals (0–5 cm, 10–20 cm, and 20–30 cm), yielding 36 field samples. Samples were freeze-dried, ground, and homogenized for spectral analysis using diffuse reflectance Fourier-transform infrared spectroscopy (FTIR-DRIFT). Data for each depth per tree were acquired in analytical triplicates, generating an FTIR-DRIFT dataset of 108 spectra. Spectral pre-processing in Quasar included rubberband baseline correction and Standard Normal Variate (SNV) normalization. Numerical peak integration was performed for bands assigned to aliphatic compounds (C–H stretching: 2830–2960 cm⁻¹), associated with labile organic structures, and aromatic/conjugated carbonyl groups (C=C ring stretching and C=O: 1590–1660 cm⁻¹), associated with chemical recalcitrance. The aromatic-to-aliphatic ratio (1590–1660 / 2830–2960 cm⁻¹) served as a proxy for SOM recalcitrance and humification. Non-parametric Kruskal-Wallis testing in RStudio indicated significant differences across soil depths (p < 0.0001). Post-hoc pairwise Mann-Whitney U tests with Bonferroni correction confirmed significant differences among all evaluated depth pairs (p < 0.001), with the largest shift in recalcitrance observed between the 0–5 cm and 20–30 cm layers (p = 1.58 × 10⁻¹¹). Median ratio values increased with depth (0.64 in 0–5 cm, 1.30 in 10–20 cm, and 1.97 in 20–30 cm), alongside higher data dispersion in deeper layers. These results indicate that while the surficial horizon retains fresh, plant-derived aliphatic carbon, subsurface layers exhibit a selective accumulation of recalcitrant aromatic structures associated with organo-mineral stabilization. This vertical differentiation suggests that after 20 years of secondary succession, the soil has recovered its architecture of functional chemical stratification, transitioning away from the homogeneous pattern typical of degraded pastures toward the stratified profile of mature tropical forest soils. In conclusion, FTIR-DRIFT analysis effectively resolves vertical shifts in SOM recalcitrance, while future applications of elemental analysis–isotope ratio mass spectrometry (EA-IRMS for bulk δ¹³C) and solid-state cross-polarization magic-angle spinning nuclear magnetic resonance (CP-MAS ¹³C NMR) can further enhance analytical certainty and structural validation.