# Metabolomic Insights into the Potential of Chestnut Biochar as a Functional Feed Ingredient
## 4 Discussion  
In this study, a mixture of woodchips and chestnut shells, which are an agricultural byproduct of chestnut production, was used to produce biochar through gasification in a Femto Gasifier system, in line with circular economy practices. The biochar was produced at temperatures exceeding 500 \({}^{\circ}\)C, which is considered suitable for animal feed. At these high temperatures, biochar retains beneficial volatile compounds while reducing harmful substances, such as polycyclic aromatic hydrocarbons (PAHs), which are more prevalent in biochar produced at lower temperatures [26]. Scanning electron microscopy (SEM) analysis of the chestnut biochar revealed a highly porous structure with fragments formed by fibers, vessels, parenchyma, and integument tissues. The presence of these tissues is coherent with the starting material used to produce biochar, such as wood and chestnut agro-residues (cupules, pericarps, and seeds). Fibers and vessels lignify the cell walls, which form the border of large channels and provide a substantial surface area for the exchange of chemicals, salts, ions, and metals. In addition, large parenchyma cells, which lack proximal cell walls, increase the binding capacity of biochar. Lignin is a complex organic polymer found in the cell walls of specific tissues, particularly in woody tissues and chestnut cupules/pericarps. Lignin-rich materials typically yield biochar with complex stable porous structures and higher pyrolysis temperatures further increase the porosity by removing volatile compounds and promoting the formation of a stable carbon matrix [27; 28]. In chestnut biochar, the larger area of vessels and fibers lumen and parenchyma broken cells provide a higher absorption capacity for both binding organic molecules and bacterial colonization. In general, the porosity depends on the feedstock and production temperature. Biochar produced at temperatures greater than 500 \({}^{\circ}\)C also has a high porosity and a large surface area, which increase its ability to adsorb toxins and gases such as ammonia in the digestive system [29]. The chestnut biochar produced was alkaline, with a pH above 10, which is consistent with previous studies showing that biochar produced at temperatures exceeding 500 \({}^{\circ}\)C tends to be alkaline, while lower production temperatures yield more acidic biochar [27]. The alkalinity of biochar is influenced by the presence of salts, such as potassium, calcium carbonates, and chlorides. The chemical composition of biochar from chestnut agro-residues was characterized by a high ash content (25.00 \(\pm\) 0.25%), reflecting the significant presence of minerals, including potassium, sodium, magnesium, and calcium. Higher pyrolysis temperatures generally lead to increased ash content as organic matter decomposes, preserving the mineral content. Figure 5: (**a**) _L. reuteri_ growth with 0, 50, and 100 \(\upmu\)L/mL of CB biochar over time; (**b**) _L. plantarum_ growth with 0, 50, and 100 \(\upmu\)L/mL of CB biochar over time. Different superscript letters indicate significant differences at \(p\) < 0.05 among different concentrations within the same time point. Heavy metal analysis of the chestnut biochar showed levels below the threshold set for use as a feed ingredient (Dir. 2002/32/EC), thus highlighting that the biochar is safe and of good quality for animal nutrition. Metabolomic analysis of the water extract revealed the presence of functional compounds, such as succinic acid, methyl coumarins, and small polyphenols. Succinic acid, which is an organic compound, is known for its strong antioxidant properties, as it acts as a metal scavenger and promotes the degradation of hydroperoxides without forming free radicals [30]. Coumarins, a class of organic compounds found in various plants, were also detected in the biochar. They are known for their diverse biological activities, including antimicrobial and anti-inflammatory properties.