# Lignocellulosic Biomass as a Substrate for Oleaginous Microorganisms: A Review
## 4 Biomass Pretreatment to Decrease Recalcitrance
### Principal Pretreatments  
#### 4.1.1 Acid Pretreatment  
Acid pretreatments are effective in breaking the lignocellulosic matrix at the glycosidic bonds between lignin and hemicelluloses, by solubilizing most of the hemicelluloses (commonly over 90% _w/w_), as well as reducing some of the cellulose and removing part of lignin (Table 1). The process consists first in the hydrolysis of hemicelluloses to its monomeric sugars, and then in the depolymerization of the cellulose into oligosaccharides, with further release of monomeric sugars. Both steps allow for a significant increase in porosity and a significant lignin removal. However, acid pretreatment is suitable for removing lignin from hardwoods and agricultural residues, but not from softwoods. The difficulty of removing lignin from softwoods is well documented and is due to the structural characteristics of softwood lignin [167; 200]. The acids used can be both inorganic (e.g., phosphoric, nitric, hydrochloric or sulphuric acid) or organic (e.g., acetic, citric, maleic or oxalic acid) [167; 186; 187], with sulphuric acid being the most commonly used. Acid pretreatment can be wet acid, using concentrated (>30% _w/v_) or diluted (<30% _w/v_) in water acid, or dry diluted acid, which has been recently documented [186; 187; 206]. Pretreatment using concentrated acid is a process that makes it possible to obtain a high content of assimilable sugars (>50%) with low energy costs since it is carried out at low temperature (<100 \({}^{\circ}\)C), even at room temperature. The disadvantages of using concentrated acids are the requirement of corrosion resistant equipment that increases investment costs and the risk of the reagents, which are highly dangerous and require investment in safety issues. Additionally, concentrated acid causes a partial degradation of hemicelluloses to by-products that accumulate in the liquid waste as organic acids, furfural and 5-hydroxymethylfurfural (HMF), mostly, reporting total values even over 13 g/L [204]. These have been reported as inhibitors of enzymatic hydrolysis and fermentation. Strategies have been developed to reduce the inhibitor concentration, such as the use of two stages acid pretreatment, where a less concentrated acid is first applied to extract hemicelluloses, causing a restricted degradation, following by a more concentrated acid, aiming the cellulose destruction [204; 216]. However, the disadvantages have made them less attractive processes to implement and, in this sense, diluted acid pretreatments are preferred. Diluted acid pretreatment is less aggressive, more environmentally friendly, and less costly, given the lower acid demand [172]. In addition, it allows the recovery of much of the sugar from hemicelluloses at the end of the process, usually with a low production of inhibitory compounds (Table 1). Most pulps obtained from diluted acid are very accessible to the enzymes; therefore, it is possible to recover glucose and a cellulose solid fraction that usually has over 70% digestibility (Table 1). The disadvantage of diluted acid pretreatments is that they require higher temperatures than those used in treatments with concentrated acids, thus increasing energy costs. The release of glucose in diluted acid pretreatments has been optimized by using more than one stage, alternating high and low temperatures during short and long retention periods [205; 217]. Solid catalysts may also be used in the pretreatment (e.g., carbon-based, zeolites, ion exchange resins) that can be recycled and reused several times. These improve the yields of assimilable sugars and leave a cellulose fraction that is easily hydrolysate, achieving glucose yields of more than 91% after enzymatic hydrolysis [187; 218]. Pretreatment with dry diluted acid has been proposed as an alternative to the use of diluted acid pretreatment. In this process, both the raw material and the product are generally solid. The biomass is even used at more than 70% _w/w_ with respect to the diluted acid, which is impregnated and fully absorbed by the biomass [206].