# Bivolarization of Grape Stalks as Animal Feed by Solid State Fermentation Using White-Rot Fungi
## 1 Introduction  
The wine industry is one of the most important agricultural activities throughout the world and produces large amounts of agro-industrial residues, representing a waste management concern, both ecological and economically [1, 2]. Grape stalks (GS) are one of those by-products, obtained by the destalking procedure just before the wine fermentation process. On average, 4 kg of GS are generated per hectoliter of produced wine [3, 4]. Recently, there is a growing interest in reducing the impact of agro-industrial wastes by using them as possible raw materials for new feed products and other applications, thus enhancing the contribution to the general policy framework inherent to the implementation of a circular economy [2, 5, 6].  
Similarly, to other lignocellulosic biomass, GS presents a low protein content (\(<\)30 g/kg DM) and a high (\(>\)50 g/kg DM) lignified cell wall content [7, 8] that limits feed intake and animal performance [9, 10]. Lignin is a phenolic compound with a high molecular weight that adds rigidity to the cell wall structure, being resistant to most microbial enzymatic systems.  
Consequently, the digestibility and availability of cell wall contents to rumen microorganisms are low [11; 12]. Among other factors, a low access of rumen microbial enzymes to structural carbohydrates is due to the linkages between lignin and cellulose and hemicellulose, hindering the availability of significant amounts of potential energy [7; 12; 13] that could otherwise be used by the microbial population in the rumen. Thus, the utilization of methodologies to break down lignin-carbohydrate linkages is essential to improve the access of rumen microbes to cellulose and hemicellulose, and consequently enhance their digestibility.  
Different treatments of lignocellulosic biomass have been studied for improving its nutritive value. In general, physical, and chemical treatments are not cost-effective and are not environmentally friendly processes. By contrast, biological treatments have been drawing more attention due to their low costs, simplicity, higher yields, and, simultaneously, their higher sustainability. Additionally, the conversion of lignocellulosic biomass into more nutritive and easily digestible products has been underlined by several authors [14; 15; 16].  
Solid state fermentation (SSF) is one of the possible biological treatments that is known to be able to reproduce the natural culture conditions of microorganisms; it can cope with substantial volumes of biomass and have a low environmental impact and reduced water consumption [17]. Many microorganisms are capable of degrading lignin by using cellulose and hemicellulose as carbon and energy sources [18]. A small group of filamentous fungi, known as white-rot fungi (WRF), has evolved in different ecological frameworks, enhancing their selective lignin-degrading ability due to their rich network of ligninolytic enzymes [18; 19]. WRF can maximize the production of lignocellulosic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and lactose. These enzymes oxidize lignin and a wide range of lignin analogous compounds and break down lignin-carbohydrate linkages [20].  
The use of WRF as a biological treatment has been widely reported [7; 21; 22; 23] as a promising, affordable, and environmentally acceptable approach, where an increase of the nutritive value of several raw materials was observed. However, changes in lignin degradation depend not only on the chemical composition of those raw materials, but also on the fungus species and incubation period used [7; 24]. In this context, the purpose of this study was to evaluate the effect of incubation periods (28, 35, and 42 days) and fungal species (_Lentiunla edodes_, _Pleurotus citrinopileatus_ and _Pleurotus erggii_) on the improvement of GS nutritive value, analyzing the effects on its chemical composition and _in vitro_ organic matter digestibility.