USE OF BIOCHAR, OBTAINED FROM THE VALORISATION OF AGRO-RESIDUAL BIOMASS, AS FUNCTIONAL FEED FOR MONOGASTRIC ANIMALS

ABSTRACT The growing global population is driving the demand for increased livestock and agricultural production, while also requiring compliance with emerging political and social mandates for sustainability and environmental protection. Addressing this challenge necessitates a transition toward a circular economy, in which waste is not discarded but instead recycled and reintegrated into production processes. This model is increasingly seen as a key solution to current global challenges related to agriculture, environmental stewardship, and resource efficiency. In this context, biochar could play a significant role. Biochar is a by-product generated alongside bio-oil and syngas through the thermochemical degradation of biomass at high temperatures (over 350°C) in an oxygen-limited environment. It has garnered significant interest for its potential to mitigate climate change through carbon sequestration and the reduction of greenhouse gas emissions. Considered a carbon-negative technology, biochar can lock carbon in the soil for centuries, thereby preventing its release into the atmosphere as CO₂. Although it is primarily recognized as a soil amendment in agriculture, biochar has recently gained attention as a potential feed additive. The properties of biochar vary considerably depending on factors such as the production technology (particularly temperature) and the type of biomass used as feedstock. As a result, biochar exhibits highly variable physical and chemical characteristics. When used as a feed additive, biochar generally provides beneficial effects without evident negative consequences. However, the scientific literature reports inconsistent results, largely due to the physical and chemical characteristics variability. The general aim of this PhD thesis was to evaluate the potential use of biochar as a functional ingredient in animal nutrition through both in vitro and in vivo approaches. In the first phase of the research, different biochar products derived from various biomass sources were characterized. Particularly, biochar samples were assessed for their chemical and metabolomic composition, microscopic structure (via SEM analysis), and key functional properties, including antioxidant activity (ABTS assay), inhibitory capacity against major Escherichia coli pathotypes, and prebiotic activity on Lactobacillus strains. To further explore possible mechanisms of action, gene expression analyses were performed using quantitative real-time PCR (qPCR), with a focus on quorum sensing pathways, particularly genes involved in biofilm formation and cell division. Regarding functional properties, biochar demonstrated a clear inhibitory effect on the growth of Escherichia coli pathotypes, primarily by modulating the expression of genes involved in quorum sensing pathways, particularly those regulating biofilm formation and cell division. Additionally, biochar showed a species-specific prebiotic activity, promoting the growth of beneficial bacterial strains without exerting any detrimental effects. The in vitro studies confirmed also the variability of the chemical and functional characteristics of biochar depending on the type of biomass used, emphasizing the importance of considering this variability when applying biochar. Overall, the results suggest a promising potential for biochar as a functional feed additive, capable of selectively inhibiting pathogenic bacteria while supporting probiotic populations, though careful consideration of the biomass source is crucial for its effective use. Finally, an in vivo trial was conducted to assess the application of biochar as a functional feed ingredient in pig nutrition. For this purpose, 223 weaned piglets were randomly assigned to two groups: a control group (CTRL) fed a standard diet, and a treatment group (TRT) fed the same diet supplemented with 1% chestnut-derived biochar. Body weight, feed intake, and fecal consistency were measured weekly, while fecal samples were collected for microbiological analysis and nutrient digestibility assessment. The TRT group showed significantly lower fecal Escherichia coli counts at 14 days and a marked reduction in diarrhea incidence at 28 days (32.14% CTRL vs. 3.23% TRT; p = 0.009). Protein digestibility was significantly higher in the TRT group (79.5 ± 1.74%) compared to CTRL (75.0 ± 2.05%; p = 0.004). Moreover, oxidative stress, assessed via reactive oxygen metabolites (d-ROMs), was significantly lower in the TRT group (293.44 ± 59.28 vs. 553.98 ± 61.59 UCARR; p ≤ 0.001). These results suggest that biochar supplementation could improve gut health and reduce oxidative stress in post-weaning piglets, supporting its potential as a sustainable functional feed ingredient in swine production. Finally, the study investigated the potential of biochar to serve as a carrier for bioactive molecules, demonstrating its promising capacity to modulate their release under varying environmental conditions. In conclusion, this research highlights the potential of biochar as a valuable feed ingredient to combat enteric disorders, reduce antibiotic reliance in livestock, and improve resource efficiency, ultimately contributing to the environmental sustainability of livestock production.