Page 16 - Read online

Page 12 of 19 Maffia et al. Carbon Footprints 2026, 5, 7

transformation of fresh organic matter into more recalcitrant and stable fractions. This mechanism is crucial
for carbon sequestration, since HSs represent the most stable pools of SOC, with residence times ranging
from decades to centuries. Compost and vermicompost treatments showed the highest values of TOC and
TEC, along with superior humification indices such as the HC/FC ratio, humification rate and HD. These
parameters indicate that a larger share of organic matter is converted into humic fractions, which are more
resistant to microbial mineralization. In particular, the highest HC/FC ratio with compost and vermicompost
reflects an enrichment of larger, more aromatic, and condensed molecules, typical of stable organic matter.
Together, these findings show that compost and vermicompost do not simply add carbon to soils, but also
transform it into more stable forms, thereby strengthening the long-term sequestration potential. Microbial
roles in carbon stabilization. The microbial community shifts observed under different treatments further
explain the pathways of carbon sequestration and humified material formation: Fungi, particularly
stimulated under digestate and SBO treatments, are central players in the decomposition of plant residues
because of their ability to degrade lignocellulosic materials through extracellular oxidative enzymes such as
laccases and peroxidases . Their filamentous growth allows them to penetrate plant tissues and initiate the
[45]
breakdown of recalcitrant organic matter, releasing soluble compounds that can be further metabolized by
other microorganisms [46,47] . Although fungi efficiently transform complex polymers into aromatic precursors,
recent research indicates that their biomass contributes less to persistent humus formation than previously
assumed. Instead, bacterial necromass represents a large and stable fraction of soil organic matter [48,49] . This
emerging view aligns fungal activity with early-stage decomposition and precursor formation, while
assigning bacteria a leading role in long-term carbon stabilization via the “microbial carbon pump”. Bacteria
- particularly enriched under vermicompost - decompose labile substrates and generate cell wall residues and
extracellular polysaccharides that bind to minerals and persist in soil. Actinomycetes, enriched in compost
and vermicompost soils, bridge fungal and bacterial functions: they degrade cellulose and hemicellulose
while also producing metabolites that serve as humus precursors. Their slow turnover further enhances
organic matter stabilization.

These complementary microbial functions reconcile seemingly conflicting findings in the literature: fungi
dominate transformation processes and generate aromatic intermediates, whereas bacteria contribute
disproportionately to humification and to mineral-associated carbon. Our results support this framework,
showing that treatments fostering diverse and active microbial communities - such as compost and
vermicompost - enhance both precursor formation and stable carbon accumulation. Long-term fertilization
studies show strong correlations between SOC and humic fraction with a coefficient of determination up of
98% , reflecting the formation of resistant aromatic-aliphatic humic structures and their stabilization
[50]
through mineral interactions [51,52] .

The microbial patterns observed here are consistent with these mechanisms: fungal activity enhances
aromatic condensation (lower E4/E6), while bacterial and actinomycete contributions promote necromass
stabilization and soil aggregation. The results indicate that compost and vermicompost represent the most
effective amendments for promoting stable carbon sequestration. By increasing humification indices and
stimulating diverse microbial communities, they foster the buildup of humic pools with slower turnover
rates. This is particularly relevant for Mediterranean agroecosystems, where climatic stressors (high
temperatures, drought) accelerate the mineralization of labile carbon pools. In contrast, digestate contributed
to SOC accumulation but with lower humification indices and microbial diversity, suggesting a more
transient carbon contribution. SBO, while stimulating fungal dominance and potentially enhancing
aggregate-associated carbon, showed limited benefits in terms of humification indices and carried a larger
environmental burden due to sulfur processing.