# Lignocellulosic Biomass as a Substrate for Oleaginous Microorganisms: A Review
## 6 Enzymatic Hydrolysis  
Enzymatic hydrolysis of lignocellulosic after pretreatment is the last step before fermentation. The most commonly used enzymes are cellulases and hemicelllases, which convert cellulose and hemicellloses into glucose and a mixture of pentoses and hexoses, respectively [243]. Additionally, mixtures of cellulases (Celluclast 1.5L, Sigma-Aldrich, St. Louis, MO, USA) and \(\beta\)-glucosidase (Novozyme 188, Sigma-Aldrich, St. Louis, MO, USA) have also been commercialized [213; 244]. Cellulases represent a group of at least 15 protein families and some subfamilies that are mainly divided into endo- and exo-glucanases types and \(\beta\)-glucosidase. Endoglucanas attack the amorphous and low crystallinity regions of cellulose leaving a reducing and a non-reducing end, exo-glucanases attack the free ends and release cellobiose molecules and \(\beta\)-glucosidase breaks the \(\beta\)-1/4-D links of cellobiose, releasing glucose molecules. On the other hand, hemicelllases carry out a complete degradation of hemicelluloses and include a diverse group of enzymes, such as endo-\(\beta\)-1.4-xylanase that produces xylo-oligosaccharides and \(\beta\)-xylanase that breaks down xylan to produce xylose. Other accessory enzymes include \(\alpha\)-glucuronidase acetylxylan esterase, ferulic acid esterase, \(\alpha\)-galactosidase and \(\alpha\)-L-arabinofuranosidase, which aid in the hydrolysis of hemicelluloses side chains [71]. The synergistic action of cellulases and hemicelluloses increases the conversion rate of cellulose and hemicelluloses into free sugars. Enzymatic hydrolysis may be inefficient due to the numerous factors that negatively affect enzyme activity. Among the factors that have been reported are the high concentration of assimilable sugars in the media, the presence of inhibitory compounds generated during chemical or physical pretreatments [245], and the long residence time, which increases non-specific and irreversible binding of enzymes with residual lignin. In general, the pretreated biomass from softwood presents more non-specific and irreversible binding of the enzymes than hardwood, due to the high residual lignin content, requiring relatively higher enzyme loads for enzymatic hydrolysis. Increased enzyme specificity and activity, along with reduced cost of enzymes, is one of the main challenges that must be faced in enzymatic hydrolysis. Currently, work is being done to increase the digestibility of cellulose and decrease non-specific functions by adding non-ionic bases or surfactants such as polyethylene glycol and sorbitol ester and polyethoxylated, Tween-80, Tween-20, dodecylbenzesulfonic acid, Triton X-100 and PEG 4000, which have proven to be highly effective [47; 71; 186; 246]. On the other hand, biotechnological solutions are continuously being proposed, e.g., genes encoding for cellulases have been cloned in fungi and yeasts, bacteria, and plants to understand their function and construct new sources for enzyme production [247; 248]. Likewise, phytopathogens, having the ability to degrade plant cell walls, such as _Fusarium verticilloides, Pycnotporus sanguineus_ and _Chrysoporthe cubensis_, are of high interest from the perspective of biotechnological production of enzymes [17]. On the other hand, simultaneous enzymatic hydrolysis (saccharification) and fermentation (SSF) seems to be a great alternative in terms of lignocellulosic material conversion that can decisively reduce the processing costs. This approach integrates the release of sugars and the biosynthesis of the microbial metabolite production in a single stage, reducing the processing time, the necessary enzymatic load, the equipment and the contamination risk and resulting in a higher product yield, compared to the separate stages [248; 249].