CNDREXEL

Chi Nguyen Department of Chemistry Drexel University, Philadelphia, PA. 19104 December 4, 2010
 * ENZYMATIC CONVERSION OF CELLULOSIC BIOMASS TO CELLULOSIC ETHANOL - A REVIEW**

Cellulose is the most abundant organic polymer on earth. It is considered an almost inexhaustible and very attractive source of raw material for fuel and chemicals. Converting cellulose from biomass into biofuels such as cellulosic ethanol is now under investigation by many scientists. Cellulases are used to break down the cellulose of plant cell walls into simple sugars. Sugars can then be fermented into ethanol as well as many other products. Producing ethanol from cellulose using cellulase enzymes will help to increase ethanol production in a more environmentally friendly way, making cellulosic biomass conversion to ethanol more desirabe. This review will focus on cellulose degradation to produce cellulosic ethanol using cellulase enzymes.
 * ABSTRACT**

Fuel ethanol can be produced not only from sugar cane, corn or starch but also from cellulosic biomass (1). Ethanol from these food sources is attractive at small scale but cannot be considered good alternative fuel sources at large scale. Cellulosic ethanol from cellulosic biomass would be a better choice. One main feature of cellulosic biomass is that it is an abundant energy feedstock. That is why converting cellulosic biomass to cellulosic ethanol has been increasingly studied over the past fifty years. Cellulosic ethanol has the potential to meet most transportation fuel needs in the United States since it depends mostly on oil for transportation. According to the United States Department of Energy, America consumes about 25% of global oil whereas it only possesses about 3% of the world’s oil reserves. Thus, increasing bio-energy supplies have become primary goal of the president’s national energy policy in the US since 2001. Having an alternative and domestic based fuel source will help minimizing our need and dependence on foreign oil, as well as generate jobs in the bio-fuel industry. It will support not only support national energy growth but also economic growth. Ethanol production from cellulosic biomass also helps to maintain energy balance. Currently, R for ethanol conversion from cellulose is 5 compared to R for ethanol conversion from corn is less than 1; with R is the ratio of energy output to energy input. The cellulose residues after ethanol production process can be burned to give thermal energy. The thermal efficiency of cellulosic ethanol conversion from biomass can reach up to 70% yield (1). And even though there are CO 2 produced during the ethanol production process, CO 2 released are circled back for photosynthesis for feedstock growth in a cellulose ethanol fuel cycle. This could result in a significant reduction in CO 2 emissions, which is great for the environment (2). As a result, cellulosic ethanol from cellulosic biomass is a very promising fuel and has captured attention of many scientists nowadays.
 * INTRODUCTION**

Cellulosic biomass is a complex composite material consisting primarily of cellulose and hemicellulose bonded to lignin in plant cell walls. About two-thirds of the dry masses of cellulosic materials are present as cellulose and hemicelluloses (3). Hemicellulose is a heterogeneous polymer consisting of different kinds of neutral sugars such as pentoses, hexoses and sugar acids (4). The cellulose microfibrils are linked with hemicelluloses to form the cellulose-hemicellulose network, which is embedded in the pectin matrix. Compared to cellulose, hemicellulose can be hydrolyzed much more easily. Lignin is an amorphous heteropolymer consisting of three different phenylpropane units connecting together by some linkages. Lignin is strong and resistant to microbial attack (3). Cellulose is the predominant component of cellulosic biomass and has formula (C 6 H 10 O 5 )n. It comprises about 33% of all plant matter and is the most abundant organic compound on Earth. Cellulose in plants is a straight chain polymer containing glucan chains having thousands of glucose residues by β-1, 4 linkages in a network of inter and intra molecular hydrogen bonding and van der Waals interaction (5). These interactions make cellulose also very resistant to biological attack. The surface of cellulose is not the same throughout. There are some regions that are highly-order crystalline and others are less-ordered amorphous regions (5). It is known that amorphous regions are more vulnerable to biological attack than crystalline one. Crystalline cellulose has a structure in that all of the atoms are located in discrete positions with respect to another. The component molecules of individual microfibrils in crystalline cellulose are packed tightly and thus prevent penetration of other molecules (6). It is believed that the crystallinity of cellulose and its association with hemicellulose and lignin are the two key challenges preventing efficient cellulose breakdown into glucose molecules.
 * UNDERSTANDING BIOMASS**

The first step of producing ethanol from cellulose includes a pretreatment phase, which helps to make cellulosic biomass more amenable to hydrolysis. At this point hydrolysis can be carried out to break down biomass polysaccharides into fermentable sugars. The final steps involve the microbial fermentation of the sugar solution to produce ethanol. Cellulose first needs to be separated from the lignin seal and the crystalline structure needs to be broken so cellulolysis steps can be followed (7). There are physical and chemical pretreatments and both of them are used to obtain high efficiency. Physical pretreatment is to decrease cellulose biomass physical size and chemical pretreatment is to break apart the chemical obstacles to make it easier for cellulose digestion. Some existing pretreatment techniques include acid hydrolysis, ammonia fiber expansion, organosolve and SPORL which stands for sulfite pretreatment to overcome recalcitrance of lignocellulose (8). Acid hydrolysis is not desirable because it produces toxic inhibitors such as furfural and hydroxymethyl furfural in lignocellulosic hydrolysate (9). Ammonia fiber expansion can be a promising pretreatment that results in no inhibitory effect (3). However, it is not as effective when the cellulose biomass such as forest biomass has high lignin content. The only two techniques that can produce more than 90% cellulose conversion for forest biomass are organosolve and SPORL. SPORL yields sugar production and is considered the most energy efficient with very low fermentation inhibitor productions (8). There are two main methods, which are acidic hydrolysis and enzymatic hydrolysis. • Acidic hydrolysis is to use acid to attack and break cellulose. This method has been used since 19th century. Both concentrated and dilute acid may be used in that dilute acid requires higher heat and higher pressure condition. The cellulose is de-crystallized and sugar can be produced. However, as mentioned previously, this acid hydrolysis produces toxic degradation products that can meddle with the fermentation process to produce ethanol. • Enzymatic hydrolysis is a newer technique and is started with a pretreatment to make cellulosic biomass more vulnerable to enzymatic digestion (10). Once the cellulose has been pretreated, cellulase enzymes are used to convert the cellulosic biomass into small glucose. Glucose can then be fermented with microorganism to produce ethanol. Enzymatic hydrolysis has advantages over acidic hydrolysis in that it avoids the problems of acid and solvent recycling and no toxic degradation products are produced in the process. Enzymatic hydrolysis is thus very environmentally friendly and is considered the most promising technology for biomass conversion at this time. Enzymatic hydrolysis is also the focus of this review in later section. Baker’s yeast (Saccharomyces cerevisiae) has been traditionally used to produce ethanol from hexoses. Recently, there have been some improvements in metabolic engineering for microorganisms used in fuel ethanol production (11). Microorganisms such as Zymomonas mobilis and Escherichia coli have been aimed via metabolic engineering besides Saccharomyces cerevisiae for cellulosic ethanol production.
 * DEGRADATION OF CELLULOSIC BIOMASS**
 * __Pretreatment__**
 * __Cellulose hydrolysis__**
 * __Microbial fermentation__**

Cellulases are enzymes that catalyze the hydrolysis of β-1, 4 glycosidic bonds of cellulose, breaking insoluble cellulose into fermentable glucose (6). There are three general types of cellulases that collaborate to break down cellulosic biomass: • Endo-β-1, 4-glucanase breaks internal bonds to disrupt the structure of cellulose and expose individual polysaccharide chains • Exo-β-1, 4-D-glucanase can access the chains from the reducing end or the non reducing end of the exposed chains and cleaves two to four units to produce tetrasaccharide or disaccharide such as cellobiose. • Cellobiase or β -glucosidase hydrolyzes cellobiose to release D-glucose units. These types of enzymes alone cannot hydrolyze complex crystalline cellulose structure efficiently but working together synergistically, they can increase the hydrolysis rates significantly. Figure 1: Cellulases and their actions during the degradation of cellulose (6)
 * CELLULASES AND STRUCTURES**

Most cellulases of fungi have a two-domain structure: catalytic domain, and carbohydrate binding module (12). A catalytic domain, (CD), contains the active site responsible for the hydrolysis reaction itself. A carbohydrate binding module, (CBM) has primary function to promote adsorption of the enzyme onto cellulose (13). For cellulose hydrolysis, the difference is that the substrate is insoluble and must absorb the enzyme prior to hydrolysis compared to most other enzymatic reactions. This absorption is supported by the CBM that anchor the CD onto the surface of cellulose through van der Waals interactions and hydrogen bonding. The CBM and CD are connected by a flexible linker, which is known to maintain separation between the CD and CBM and also to provide some flexibility for autonomous function on the surface of cellulose (14). There are also multiple combinations of domains which lead to the complex structure of cellulolytic enzymes. Cellulases can be grouped into families according to sequence similarities of amino acid within their catalytic domain and carbohydrate binding module (15).

One of the most critical aspects of converting biomass to ethanol is enzymatic degradation of biomass. Even though there are extensive amounts of research over the past fifty years, knowledge of the actions of cellulase on cellulose is still far from complete (16). Enzymatic hydrolysis is a very complex system. It is proposed that the enzymatic hydrolysis of crystalline cellulose is initiated with the adsorption of cellulase to the surface of cellulose in a fast equilibrium. Then a non-hydrolytic disruption of the crystalline network of the cellulose surface to expose glucan chains slowly. The single solvated chain of cellulose now can be extracted into the active site of cellulase to form a pseudo-Michaelis complex. The pseudo-Michaelis complex is then subjected to hydrolytic cleavage to form the hydrolysis product (17).
 * ENZYMATIC HYDROLYSIS**

__**Adsorption of cellulase to cellulose**__ Adsorption of cellulase to cellulose is an important step of the hydrolytic process and is fast compared to the time required for product hydrolysis (15). It is recognized that some enzymes adsorb more tightly than others and are considered to be more efficient for the hydrolysis of cellulose (18). The adsorption of the enzymes was reversible partially (19). And as mentioned previously, carbohydrate binding modules are responsible for the adsorption of cellulase enzyme to accessible sites of insoluble cellulose (13). Formation of enzyme-cellulose complexes is a prerequisite for cellulose hydrolysis. Langmuir isotherm is the most frequent equation used to describe cellulase adsorption (20): where Ea is adsorbed celllulase (mg or µmol cellulase/L), Wmax is the maximum cellulase adsorption = Amax .S (mg or µmol cellulase/L), Amax is the maximum cellulase adsorption per g cellulose (mg or µmol cellulase/g cellulose), S is cellulose concentration (g cellulose/L), Ef is free cellulase (mg or µmol cellulase/L) and Kp is dissociation constant (L/g cellulose). The Langmuir equation is mainly used since it presents a good fit to the data in most cases and it signifies a simple mechanistic model that can be used to evaluate kinetic properties of various cellulase and the cellulose system.

__**Hydrolytic cleavage mechanism**__ For the last step in enzymatic hydrolysis, cellulase must hydrolyze the glycosidic bonds connecting the β-D-glycosyl residues of cellulose to get the product. The two key mechanisms of cellulases, the retention and the inversion glycosidases mechanism, have been studied in great detail. The mechanism of cellulases was first proposed by Koshland in 1953 then by Sinnott in 1990 (21). In most cases, the hydrolysis of the glycosidic bond is performed by two catalytic residues of the enzyme: a general acid, proton donor and a nucleophile/base. Retention or inversion at the anomeric configuration is depending upon the spatial position of these catalytic residues. Retention glycosidases have two major step mechanisms, with inversion in each step that results in a net retention of stereochemistry. Two catalytic residues of the enzyme which are usually the carboxylates group of the enzyme are involved. One residue works as a nucleophile and the other works as an acid or base depending on the step. In the first sequence, a glycosyl enzyme intermediate is formed when the nucleophile tackles the anomeric centre with acidic support provided by the acidic carboxylate. In the second sequence, the deprotonated acidic carboxylate now serves as a base and help the nucleophilic water to hydrolyze the glycosyl enzyme intermediate to finally achieve the hydrolyzed product (15). Inversion glycosidases results in an inversion at the anomeric center through single nucleophilic displacement. Hydrolysis of a beta-glycosidic bond thus results in a product with the alpha-configuration. The distance between the two carboxylates is a bit larger in inversion glycosidases mechanism compared to retention glycosidases mechanism. This inversion mechanism also involves two catalytic carboxylates but the difference is only in one step, one catalytic carboxylate presents general acid-catalysed leaving group departure and the other one presents general base-assistance to nucleophilic attacks by a water molecule. These two reacts at the same time on the opposite side of the sugar ring and produce the hydrolyzed product as shown in figure 2.

Figure 2: Inversion glycosidases mechanism (15) The mechanism of hydrolysis is conserved within a family. However, why certain families of cellulase use one mechanism and not the other one is still not known.

__**Non-hydrolytic enzymatic processes of cellulase**__ Even though adsorption and mechanism of hydrolytic cleavage to form the hydrolysis product has been studied a great deal, little success has been achieved for the fundamental knowledge of the non-hydrolytic enzymatic processes of cellulase that involved the disruption of the surface structure of cellulose and extraction of the liberated cellulose by cellulase. One of these processes has been speculated to be the rate-limiting step for the entire enzymatic hydrolysis of crystalline cellulose (16). Lacking of instruments and experimental approaches to study the interaction at the interface of insoluble cellulose and soluble cellulase heterogeneously system has made this non-hydrolytic step remains unverified.

__**Challenges**__ So far neither the rate limiting step for enzymatic hydrolysis of crystalline cellulose nor the proposal of the non-hydrolytic processes has been experimentally confirmed. Currently the efficiency of the wild type cellulases is not sufficient enough for a large feasible production of cellulosic ethanol. If new mutation features were incorporated into the protein that could speed up the rate-limiting step, the biomass conversion efficiency could be increased tremendously. To improve cellulase enzyme either naturally or genetically modified it, a comprehensive understanding of cellulase actions on cellulose needs to be achieved. It is also necessary to improve all steps in degradation of cellulose biomass including pretreatment, enzymatic hydrolysis then microbial fermentation. The focus is still suggested to be on enzymatic hydrolysis, where one of the fundamental knowledge is still lacking for the time being. It is neccessary to come up with an appropriate technique and approach to study the non-hydrolytic step. But in all, improvement in any step can advance the whole process and that will be beneficial to cellulosic ethanol production.

The energy source is a very important factor to determine the potential alternative fuel. Cellulosic biomass is not only an abundant energy feedstock but also a renewable nonfood source. Cellulosic ethanol from cellulosic biomass has tremendous potential because of its widespread availability, relative low cost of cellulosic material and it is also very environmental friendly. However, this is a rather new method and so cellulosic ethanol conversion still needs more research and attention of many more scientists. Experiments need to be carried out to obtain efficient cellulosic ethanol production with a cost as low as possible. It is important to figure out the non-hydrolytic enzymatic processes of cellulase to disrupt the surface and the extraction of cellulose. Once there is a fundamental understanding of how the cellulase works on the cellulose overall, a highly efficient cellulase for cellulosic biomass conversion can expectantly to be obtained in the future.
 * CONCLUSION**

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