# Lignocellulosic Biomass as a Substrate for Oleaginous Microorganisms: A Review
## 4 Biomass Pretreatment to Decrease Recalcitrance  
To obtain assimilable sugars from lignocellulosic biomass, it is necessary to apply previous processes that include mechanical methods, physicochemical pretreatments and enzymatic hydrolysis. First, it is necessary to reduce the size of the particles in order to decrease the degree of polymerization and to increase the surface area and porosity of the biomass to improve the exposure to the action of reagents. This can be done through a mechanical process, considered a fundamental step of the lignocellulosic biorefinery, which includes processes such as cutting or grinding (the size varies from meters to centimeters), coarse grinding (from cm to 500 \(\upmu\)m), intermediate micromization (from cm to 100 \(\upmu\)m) and fine grinding (<100 \(\upmu\)m). These are generally obtained by compression friction or shearing and therefore generate a specific energy requirement (SER) that must be evaluated [71; 181; 182; 71]. SER in mechanical treatment is affected by the moisture content and chemical composition of the substrate and has been demonstrated to increase with increasing moisture and arabinose/xylose content [183]. It has been reported that mechanical processes can increase carbohydrate production, improving yield and enzymatic hydrolysis rate by 5-25% and 23-59%, respectively, compared to untreated biomass [71]. After the mechanical process, a pretreatment is applied that leads to the reduction of the biomass recalcitrance and, in some cases, to the hydrolysis of a significant percentage of polysaccharides. However, mechanical processes are usually intended to increase the accessible surface area of cellulose and hemicelluloses to enzymes to be used in enzymatic hydrolysis [15]. Pretreatments vary and have been widely studied in productive, technical, economic and environmental terms [184; 185; 186; 187; 188]. In this context, chemical pretreatments such as acid, alkaline and acid combined with alkaline have been the most preferred and patented techniques in recent years, compared to other methods. These represent advantages associated with the use of commonly used compounds, flexibility of application at the industrial level and high final yields of assimilable sugars [171; 172; 186; 187]. Other recommended pretreatments are steam explosion and treatment with organosolv. Steam explosion (or autohydrolysis used as a synonym), is the most widely used physicochemical pretreatment method. It has been described as an economical and environmentally friendly method that reduces the concentration of toxic compounds in hydrolyses compared to other pretreatments, such as acid pretreatment [48; 186; 189]. Organosolv pretreatment, although more expensive than the other processes, may provide some valuable by-products that allow the sustainability of the process. In a bioreinery, organosolv pretreatment is one of the best options due to very good quality of cellulose obtained, very accessible to enzymes, and lignin residual from which it is possible to produces more than one valuable product (e.g., bioethanol and lignin products) in the same process [163; 190]. The selection of the pretreatment method and its operating conditions, are fundamental for a successful enzymatic hydrolysis and subsequent fermentation. The conditions used largely depend on the nature and chemical composition of the substrate (hardwood, softwood or herbaceous) and determine the amount of the generated by-products that can interfere with enzymatic hydrolysis and fermentation. Below is a description of the advantages and disadvantages of the most recommended processes (acid, alkaline, acid-alkaline, steam explosion and organosolv) and a discussion of the yields and effects of these pretreatments on enzymatic hydrolysis. Table 1 shows the most frequent values of the working conditions and yields of the different pretreatments applied to different lignocellulosic substrates.