Materials built from simple sugar molecules are the foundation of plant systems. Many plants, like corn, use these sugar-based materials to store energy in the form of starch. We then use these materials for food and increasingly as fuels and chemicals. Simple sugars are also used in structural plant materials that humans cannot digest. Some organisms, like fungi and termites, have the proper enzymes, commonly referred to as cellulases, to free up the simple sugars in these fibrous materials. The emerging cellulosic biofuels industry is spurring research and commercial efforts to improve and refine these cellulase enzymes.
Cellulases are enzymes that break down (hydrolyze) cellulose into simple sugars. Cellulose is an abundant structural molecule found in most plants and composed of linked carbohydrates. Cellulases are an essential tool used in the NARA conversion process. When added to pretreated wood, they bind to cellulose and break it into sugar molecules. This cleaving process is called “enzymatically hydrolyze” and is essential to the production of sugars that will be later fermented into the isobutanol alcohol used to make biojet fuel.
Essentially, high sugar yields from wood depends on proper conditions to aid the cellulase enzyme. But getting cellulases to perform their task efficiently is a real challenge. First, lignin and hemicellulose molecules surround the cellulose in wood and effectively block cellulases from binding to cellulose. To partially alleviate this blockage and make the cellulose accessible to the cellulase enzymes, a pretreatment step is required. Second, even if the pretreatment process exposes the cellulose, cellulase can nonspecifically bind to non-sugar molecules like lignin, effectively preventing them from functioning.
Nonspecific Binding to Lignin
One strategy used to relieve the effects of cellulases binding to lignin is to simply increase the amount of cellulase. This option works fine, however, purchasing or processing cellulase is expensive. Other strategies involve removing the lignin by extensive washing or chemically treating the lignin molecule so that cellulase does not bind. These options can also be expensive and environmentally unsound.
Elevate the pH
Work partially funded by NARA and conducted at the USDA Forest Products Laboratory, a NARA affiliate organization, provides an elegant and inexpensive way to reduce the nonspecific binding of cellulase to lignin and thereby increase the simple sugar yields from hydrolysis. NARA researcher Junyong Zhu, scientific team leader at the USDA Forest Products Laboratory and adjunct professor at the University of Wisconsin-Madison, and his collegues published two papers that describe their findings (see references below).
Traditionally, cellulose hydrolysis using cellulase is performed in solution at a pH range of 4.8 to 5.0. At this pH range, cellulases break down (hydrolyze) pure cellulose at the fastest rate. This is also the pH range recommended by Novazymes, the manufacturer of the cellulase/hemicellulase mix commonly used for cellulose hydrolysis.
What the team at the USDA Forest Products Laboratory discovered is that by raising the pH of the solution to the range of 5.5 to 6.0, they could improve the simple sugar yields from cellulose hydrolysis by up to 70%. A key consideration in this case is that the cellulose contained in wood residuals after pretreatment is not pure.
“These results contradict the well-established concept that an optimal pH is 4.8–5.0 for enzymatic hydrolysis using Trichoderma reesi cellulase”, says Dr. Zhu. “This pH range is based on optimization by using pure cellulose and is widely accepted and exclusively practiced in numerous laboratories throughout the world.”
The higher pH reduces the rate of cellulase activity, but it also dramatically reduces the nonspecific binding of cellulase to lignin, thereby providing a net improvement of cellulase activity. It is believed that the higher pH conditions alter the charge of the lignin and cellulase surface and thus reduce their binding affinity to each other.
Raising the pH in the system is relatively inexpensive and has the added benefit of placing the hydrolyzed carbohydrates in a pH range that is better suited for fermentation into isobutanol. These findings have been incorporated into the protocols currently in place to develop large-scale conversion of wood residues to biojet fuel.
Lan, T.Q., Lou, H., J.Y. Zhu. (2012) Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2-6.2. Bioenerg. Res. 6, 476-485. doi:10.1007/s12155-012-9273-4.
Lou, H., Zhu, J.Y., Lan, T.Q., lai, H., Qui, X. (2013) pH-Induced Lignin Surface Modification to Reduce Nonspecific Cellulase Binding and Enhance Enzymatic Saccharification of Lignocelluloses. ChemPubSoc. 6, 919-927. DOI: 10.1002/ceec.201200859