# Bivolarization of Grape Stalks as Animal Feed by Solid State Fermentation Using White-Rot Fungi
## 3 Results and Discussion
& 0.179 [FOOTNOTE:7]Footnote 7: Values with different letters within a column are significantly (\(p<0.05\)) different. & 0.463 [FOOTNOTE:7]Footnote 7: Values with different letters within a column are significantly (\(p<0.05\)) different. production of enzymes involved in ligninolysis and hemicellulolysis, that increased the delignification process. Changes within the structure of the cell wall of GS were also observed microscopically, by analyzing the surface morphology and micro-morphological structures of GS by scanning electron microscopy (SEM) (Figure 4). The autoclaved GS (Figure 4(a1)) showed a tight vascular structure, and the surface was relatively smooth and compact without any dumps, while the surface of the treated GS (Figure 4(b1)) was partially damaged by fungi, showing that the dense structure was loosened and became more permeable, and it is possible to visualize a darker structure in the center of the stalk, possibly a structure belonging to the xylem channels. Magnified images show that in the untreated GS (Figure 4(a2)), a cellular organization pattern with sets of long horizontal cells alternated by rounded and shorter cells can be observed. On the other hand, in the treated GS (Figure 4(b2)), images show that the organization is no longer visible, and it is possible to observe the degradation of the cell wall, showing that the GS no longer has a complete fiber structure. Data presented in this study indicated that during the treatment with WRF, lignin contents generally decrease with the increase of the IP, and that is corroborated in studies published by other authors [7, 54, 55]. One of the reasons for longer incubation periods is the potential outcome of higher delignification, as lignin is not potentially degraded during primary fungal growth due to the presence of other lignocellulosic components that limit the physical contact between enzymes and lignin [55]. However, the structural effects that WRF can enhance in the substrates depend  
Figure 3: Principal component analysis (PCA) of fungal and time incubation influence on the analyzed parameters in treated GS (Lacc, lacase; MnP, manganese peroxidase; LnP, lignin peroxidase; CMCase, carboxymethyl cellulase; FAEs, feruloyl esterases; CP, crude protein; NDFom, neutral detergent fiber; ADFom, acid detergent fiber; HC, hemicellulose; Cel, cellulose; IVOMD, _in vitro_ organic matter digestibility; C/L, cellulose/lignin (sa) ratio; LE, _L. edodes_; PC, _P. citrinopilatatus_; PE, _P. ergusii_; 28, 35 and 42 days). on the characteristics of the substrate, the fungal strain, and the incubation period [22, 35]. It should also be mentioned that the availability of soluble nutrients during the initial stages of incubation might enhance the utilization of these nutrients for mycelium growth, and only after the synthesis of extracellular enzymes will they be stimulated. In this way, longer incubation periods may reflect the fact that nutritional scarcity may trigger the synthesis of ligninolytic enzymes [56]. In this way, longer incubation periods are necessary to better understand the correlation between delignification and digestive utilization and to observe a potentially more pronounced beneficial association between them. In addition, future animal performance studies should be performed to evaluate the potential of including treated GS in animal feeding.