Hydrolysis is generally defined as the depolymeriza­tion of a substance via hydration. An aqueous acid’s ions act to cleave long polymers like cellulose, hemicellulose and lignin into smaller chains. Pretreating LB to undergo hydrolysis or converting polysaccharides into monosac­charides will enhance later fermentation by improving
the ability of anaerobic organisms to digest the resultant, simpler sugars. Hydrolysis requires extended residence time. Unfortunately, monosaccharides degrade into other nonsugar molecules when subjected for extended times to relatively high temperatures and acid condi­tions (Hsu, 1996; Wyman et al., 2005). The hydrolysis reaction rate accelerates when either a chemical or an enzymatic catalyst is used and when the material to by hydrolyzed is concentrated.

Enzyme hydrolysis is highly specific and relatively fast. Using an enzyme to act on its target polysaccharide will convert it rapidly into its component monomers. Additionally, this will convert the insoluble polysaccha­ride into a soluble monomer. Enzymatic hydrolysis is best applied after other pretreatment methods that leave cellulose as a major component. The most common method of saccharification is enzymatic hydrolysis following acid hydrolysis (Harun, 2010).

The enzyme cellulase converts cellulose into glucose. Cellulases are so specific that they only affect cellulose and do not treat hemicelluloses in the LB (Wang et al.,

2012) . There are five general types of cellulases. They are classified by the reactions they catalyze. These five cellulases are endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose phosphorylases (Bayer et al., 1998). Table 27.6 summarizes the effectiveness on the hydrolysis of wheat straw of Cellulase, alpha — Glucosidase and Xylananse from T. reesei, A. niger, and T. longibrochiatum after various pre-treatments. The high yields and mild conditions are attractive for commercial applications.

These enzyme structures are complex and can be found in various bacteria as organized supramolecular complexes called cellulosomes (Bayer et al., 1998). These enzymes are commonly found in fungi such as Tricho — derma reesei and Aspergillus niger and in bacteria such as Clostridium cellulovorans (Arai et al., 2006). These source organisms are either aerobic or anaerobic and

are either mesophilic or thermophilic. Commercial pro­duction of cellulase is focused on fungal sources because bacterial sources tend to be anaerobic and thus are slow to grow (Duff and Murray, 1996).

It appears that at least three classes of enzymes act together, synergistically, to hydrolyze cellulose: endocel — lulase, exocellulase, and cellobiase. Endocellulase (EC

3.2.1.4) randomly breaks internal (b-D-1,4) bonds at amorphous sites that create new chain ends. Exocellu- lase (EC 3.2.1.91) cleaves two to four units from the ends of the exposed chains produced by the endocellu — lase and results in tetrasaccharides or disaccharides. Lastly, the cellobiase (EC 3.2.1.21), otherwise known as b-glucosidase, hydrolyzes the exocellulase products into individual monosaccharides (Coughlan and Ljung — dahl, 1988; Galbe and Zacchi, 2002; Rabinovich et al., 2002; Zhang et al., 2006).

The cellulase action occurs in three steps. The first is adsorption of cellulase onto the surface of the cellulose. The second is biodegradation of cellulose into ferment­able sugars. Lastly, desorption of cellulase occurs completing the catalytic cycle.

Enzyme activity is affected by a variety of environ­mental and substantive conditions. Temperature and pH are known to affect enzyme activity. Most cellulose enzymes show an optimum activity at temperatures in the range of 45—55 °C and at pH values between 4 and 5 (Galbe and Zacchi, 2002). For LB applications, the optimum pH is shifted upward to between 5 and 6.5 due to the presence of lignin in the system (Lucas et al., 2012). These are mild operational conditions. These mild conditions lower the overall operational costs compared to purely chemical hydrolysis methods.

Additionally, substrate concentration, product con­centration, activators, inhibitors and cellulose structure are also significant determiners of enzyme effectiveness (Detroy and Julian, 1982).

Cellobiase is itself an inhibitor to endo — and exocellu — lases. Thus, the b-glucosidase activity is crucial for the efficiency of the hydrolysis process (Coughlan and Ljungdahl, 1988; Galbe and Zacchi, 2002; Rabinovich et al., 2002; Zhang et al., 2006).

The structure of cellulose affects the rate of hydroly­sis. The cellulose features known to affect the rate of hy­drolysis include (1) molecular structure of cellulose, (2) crystallinity of cellulose, (3) surface area of cellulose fiber, (4) degree of swelling of cellulose fiber, (5) DP, and (6) associated lignin or other materials (Det­roy and Julian, 1982). The purer and more refined the cellulose is, the more ideal the cellulase activity will be. Higher enzyme activity lowers the enzyme load and cost for the enzymatic hydrolysis process.

Lastly, even under ideal conditions, the activity of the cellulase enzyme is affected by the age of the enzyme it­self. The overall activity of the enzyme decreases rapidly and slows the rate of enzymatic hydrolysis. There is currently much research devoted to improving the over­all yield and maintaining a high rate of hydrolysis (Sun and Cheng, 2002).

Supplementing cellulase enzymes with other en­zymes is another area of current focus. Conjugating the action of cellulases and hemicellulases is known to increase the rate of enzymatic hydrolysis and result in an overall higher sugar yield. Cellulose is a homopoly­saccharide, hemicelluloses are heteropolysaccharides. To obtain a more complete hydrolysis of LB one must consider a multiple-enzyme system and reap the yield of the combined activities.