**4. Pretreatment of lignocellulosic biomass**

## **4.1 Objectives of pretreatment and basic methods**

Without pretreatment before the enzymatic saccharification stage, the nonbiodegradable lignin in lignocellulosic material presents as a major obstacle to the enzymatic hydrolysis of crystalline cellulose and hemicellulose which themselves already have low digestibility [48]. Pretreatment removes or decomposes the lignin (delignification) [49] and thus makes cellulose and hemicellulose more readily available to cellulases and hemicellulose's.

In principle, there are three methods for pretreatment: biological, chemical and physical processes. Some processes, where chemical and physical actions are inherently inseparable, are termed physiochemical. Two or all of these basic methods can be used in combination to gain benefits from each method. Various pretreatment methods have been described and compared critically in a recent review [50].

Biological treatment uses microorganisms such as white, brown or soft rot fungi which break up the structure of lignin via the action of extracellular lignolytic enzymes released by the fungi [51]. Further research is needed to overcome the issues of selectivity, cost, retention time and effectiveness to make it a practical choice [50].

Chemical treatments include treatment with bases, diluted acids, and oxygen as an oxidizer. These reagents react with lignin and cause the polymer to breakdown into smaller and more soluble fragments. Physical pretreatment is usually performed before chemical or biological treatment to reduces cell wall crystallinity and particle size by physical milling or grinding [50]. In some treatment methods, both physical action and chemical reaction play important roles in lignin removal. Such physicochemical pretreatment can involve steam explosion, liquid hot water, ammonia fiber explosion, ammonia recycle percolation or a supercritical carbon dioxide.

Pretreatment contributes a vital role in the cost evaluation process of whole technology, because they contribute about 30–35% of overall production cost [52]. There are many issues that arise from this process [50] including loss of sugars (mainly pentose sugars derived from hemicellulose degradation), and generation of toxic substances that inhibits the downstream fermentation process. Both need to be minimized to make ethanol production more efficient.

## **4.2 Steam explosion**

Steam explosion has become one of the most adopted pretreatment processes, where hydrolysis of hemicellulose also happens which improves cellulose digestibility. It is a physiochemical method that uses both physical changes caused by sudden pressure reduction and heat- and catalyst-induced chemical changes. An impregnation agent is sometimes used before the pretreatment step. Upon steam explosion after 1–5 min soaking in 160–270°C and 20–50 bar steam, fibers loose up and sugar polymers (mainly hemicellulose) partially degrade into sugars via hydrolysis of glycoside bonds in polysaccharides and lignin into soluble fragments including some inhibitors and phenolic products [50]. The process allows for subsequent solubilization of hemicellulose in water and lignin in organic or alkaline solvent. Cellulose undergoes some degree of polymerization but is still insoluble in water or organic solvents and remains in the solid phase. Acid (sulfuric acid and sulfur dioxide) impregnation before steam explosion reduce the time and temperature necessary for proper depolymerization of the feedstock, increases the efficiency of enzymatic hydrolysis of polysaccharides to glucose and xylose and reduce enzyme consumption [53]. Compared to other methods of biomass fractionation, steam explosion uses less dangerous chemicals, less demanding on investment and energy consumption [54]. Steam explosion is not recommended for agricultural and hardwood wastes with high contents of pentoses and low levels of lignin, due to the susceptibility of pentoses to thermal degradation. Steam explosion is recommended for processing straw and bagasse.

#### **4.3 Inhibitors generated in pretreatment**

One of the lasting issues in the second-generation bioethanol production is the formation of inhibitors during the pretreatment. The inhibitors create unfriendly environments for fermentative microbes, increases the length of lag phase, causes loss of cell density and lower growth rates of fermenting microbes, and consequently decreases ethanol yields [55]. The commonly observed inhibitors are aldehydes such as 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde (furfural), weak organic acids (formic, acetic and levulinic acids) and phenolic compounds [56]. Acetic acid is the major organic acid found in hydrolysates coming from the

hydrolysis of acetyl side-chain groups in hemicellulose [57]. Cell growth of fermentative microbes is inhibited by the intracellular process of anions of weak acids. Furan aldehydes are poisonous for microbes and phenolic compounds interfere with the function and integrity of cell membranes [58].

There are several methods used for the removal of inhibitors [59]. The detoxification of lignocellulosic hydrolysates can be performed using inhibitor sorbents such as excess of lime, active carbon or lignite (brown coal).
