# Metabolomic Insights into the Potential of Chestnut Biochar as a Functional Feed Ingredient
## 3 Results
### Morphological Characterization  
The morphology of the biochar was examined by SEM. At low magnification (Figure 2A), a mixture of small and large fragments was observed. However, the overall texture of the sample consisted mostly of fragments with dimensions of approximately 100-300 \(\upmu\)m (considering the largest fragment coordinates). Only a few materials had dimensions of 1 mm or as small as 50 \(\upmu\)m. Many of these fragments had retained the structural characteristics of the original plant tissues. Many aggregations of vascular and fibrous cells (Figure 2B-E) were found, probably derived from chestnut wood or vascular bundles of fruit remains. The lignin in the cell wall led to good preservation of these structures.  
The tracheids were well structured (Figure 2B,C), with smooth cell walls, characterized by oval pits (taxodioid; Figure 2C, arrows). The shape and organization of these pits confirmed that they belonged to chestnut wood [25]. Fibers appeared as closely packed cells with a regular diameter (Figure 2D,E), sometimes flattened by treatment (Figure 2D, asterisk), with smooth cell walls. Occasionally, debris, probably derived from the breakage of cells from living tissue (parenchymal cells), filled the cell cavities of these fibers, blocking their lumen (Figure 2E, arrows). The average lumen area of these cells was 27 \(\upmu\)m\({}^{2}\), while the average lumen area of the thinner end of the tapered cells was 1.5 \(\upmu\)m\({}^{2}\).  
In addition to lignified cells, other different tissues were recognizable in the fragments. The integumentary tissues showed neighboring cells with no intercellular space (Figure 2F). Some fractures were visible along the middle lamella (Figure 2F, arrow) and the proximal cell wall of some cells had disappeared (Figure 2F, asterisk), probably due to the sample preparation. Parenchymal tissues were characterized by large cells with a main dimension of 50 \(\upmu\)m and an area of one mm\({}^{2}\) (Figure 2G, asterisk). The fragment in Figure 2G shows parenchymal cells surrounding a fiber aggregate (Figure 2G, arrow). The cells on the surface of the fragment lacked the proximal cell wall and contained cell debris (Figure 2G). Debris lost during cell treatment had aggregated to form fragments of amorphous material in which plant tissue was not observed (Figure 2D, arrows).  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Mineral** & **Content (g kg\({}^{-1}\))** & **Mineral** & **Content (mg kg\({}^{-1}\))** & **Mineral** & **Content (mg kg\({}^{-1}\))** \\ \hline Na & 10.34 \(\pm\) 1.70 & Cr & 66.56 \(\pm\) 8.22 & Zn & 1477.47 \(\pm\) 321.71 \\ \hline Mg & 7.36 \(\pm\) 1.16 & Mn & 721.99 \(\pm\) 170 & As & 2.06 \(\pm\) 0.41 \\ \hline Al & 2.74 \(\pm\) 0.11 & Fe & 6720 \(\pm\) 0.61 & Se & 0.15 \(\pm\) 0.05 \\ \hline P & 3.34 \(\pm\) 0.62 & Co & 1.93 \(\pm\) 0.51 & Mo & 2.38 \(\pm\) 1.59 \\ \hline K & 33.56 \(\pm\) 3.06 & Ni & 28.72 \(\pm\) 6.66 & Cd & 0.20 \(\pm\) 0.04 \\ \hline Ca & 9.31 \(\pm\) 1.09 & Cu & 59.89 \(\pm\) 1.50 & Pb & 8.05 \(\pm\) 0.39 \\ \hline \end{tabular}
\end{table}
Table 2: Mineral content of CB biochar by ICP-MS. The results are expressed as the mean \(\pm\) sd (\(n\) = 3).  
Figure 2: Morphological analysis of biochar from chestnut agro-residue biomass by scanning electron microscope (SEM). (**A**) Image at low magnification; (**B–G**) Image at high magnification.