Cellulose Degradation

In a simplistic summary, there are four major steps in generating bioethanol and other biomass-derived products: (a) generate biomass through photosynthesis, (b) process raw biomass to a form suitable for microbial fermentation, (c) ferment biomass into ethanol (or other products), and (d) recover product and recycle unfermented residual biomass. We will not dwell here on the first step that takes place in nature. We present the remaining steps to place in proper perspective fungi based cellulose degradation. Pretreatment of the cellulosic biomass is necessary in the yeast and bacteria based cellulose conversion, because these microorganisms do not easily hydrolyze woods and agricultural residues. If they did, biomass material would not remain for long nor accumulate (and wood would be totally unsuitable as a

Table 3 Optimum pH and temperature range


Optimum pH Optimum temperature (°C)

F. oxysporum F. oxysporum F. oxysporum Monilia sp. Mucor sp. N. crassa

30 30 34 26 30


building material). In order for organisms to digest biomass efficiently and productively, it must be pretreated by mechanical, chemical, or biological means to break down the stable polymeric structure of cellulose, hemicellulose, and lignin. For this purpose, there are presently four major upstream processing technology platforms, and many variations exist around these basic technologies: (a) concentrated acid hydrolysis carried out at low temperature, (b) dilute acid hydrolysis carried out at high temperature, (c) enzymatic (cellulase) hydrolysis, and (d) biomass gasification (pyrolysis). The two acid technologies are old developments; whereas, the enzymatic process is relatively newer, and the gasification process is much more recent. Enzymatic hydrolysis alone is normally insufficient in degrading cellulose; thus, mechanical and chemical pretreatment becomes necessary. Finally, the fermentation step normally is further subdivided into two steps: (a) conversion of cellulose to glucose and (b) conversion of glucose to ethanol (Blazej and Kosik 1985). A separate organism is usually responsible for each one of these two steps. Only a few microorganisms in nature can perform tasks in both steps.

Physical pretreatments can be either mechanical or nonmechanical (Fan et al. 1987). In the mechanical process, the force applied in pretreatment breaks lignocellulosic materials into small highly digestible particles for enzymatic hydrolysis, where soluble enzyme works on insoluble cellulose mainly at the liquid-solid interface and the observed reaction rate is not necessarily limited by the intrinsic reaction rate but by mass transfer. Thus, breaking down particles increases the surface area for reaction. In addition, the nonmechanical pretreatment with high temperature steam causes disruption in the fundamental molecular and crystalline structure of the cellulose.

Ball Milling: Ball milling is one of the best methods to enhance enzymatic hydrolysis. In this process, shear and mechanical forces break down the particle size in order to facilitate the digestion of cellulose. Ball milling, although highly efficient, cannot be operated on a large scale because of the high cost and the lengthy time it takes to break down the molecular structure of cellulose. Like ball milling, two-roll milling also decreases the particle size.

To facilitate digestion, chemical agents such as alkali or acid solutions have long been utilized to pretreat cellulose. Acid treatment increases the reduction power of the reactive groups along the cellulose polymer chain, and cellulose is thus destabilized and rendered more susceptible to subsequent attacks (Fan et al. 1987). Concentrated acid disrupts the hydrogen bonds between cellulose chains, thus, it breaks the otherwise highly stable crystalline structure of cellulose and dissolves cellulose into a thick, hydrogel-like solution. Cellulose is now in an amorphous state and is readily digested. Addition of water dilutes the acid and rapidly breaks the b-1,4-glucosidic linkage, leading to complete hydrolysis of cellulose to glucose (known as saccharification) with an yield that is close to the theoretical value. At the end of saccharification, glucose molecules remain intact. Concentrated sulfuric acid is the acid of choice, although other acids

(hydrochloric, nitric, and phosphoric) are also employed in some laboratory studies. In a typical process, five parts of ~ 75% sulfuric acid is added to four parts of dried biomass containing ~ 10% moisture at 50°C. This is followed by diluting with water to ~ 25% acid and holding at 100°C for 1 h. The following schematic shows the fate of cellulose in concentrated acid.

cellulose ! acid complex ! oligosaccharides ! glucose

High temperature dilute acid process works in two stages, with the first stage optimized for the more readily hydrolyzed hemicellulose and the second stage's conditions specifically tuned for the tougher cellulose. Typical conditions are 0.7% sulfuric acid at 190°C for the first stage and 0.4% at 215°C for the second stage. The residence time for each stage is approximately 3 min. A dilute acid process exposes cellulose to acid for a shorter time compared to a concentrated acid process, but only about 50% of the glucose is recovered in a dilute acid process compared to nearly 100% recovery for a concentrated acid process. In hot dilute acids, hydrolysis of cellulose proceeds as follows.

native cellulose ! stable cellulose (hydrocellulose) ! soluble polysaccharides ! glucose

Under certain conditions, alkaline solutions can degrade cellulose by breaking down its long molecular chain at random interchain locations and generate a number of shorter molecules of cellulose. Many alkalis such as metal hydroxides, salts in a strong alkali solution, inorganic salts, a number of amines, and related compounds break the hydrogen bonds between cellulose molecules. Alkalis commonly employed in practice include sodium hydroxide and ammonia. The cellulose structure resulting from alkaline treatment is more amorphous. The swollen cellulose structure allows subsequent enzymatic degradation to proceed more readily because enzyme molecules can gain access to its interior region. Unlike acid treatment, alkaline treatment alone is usually insufficient in releasing constituent monomer sugars to theoretical yield. As a result, it is normally only a pretreatment step that is followed by an enzyme step. Table 4 shows the sugar yields from different alkali pretreated biomass sources after enzymatic digestion.

Biological degradation of cellulose features prominently in the nature's carbon cycle. Cellulase refers generally to the enzymes that degrade cellulose. Most known cellulases are of fungal origin. Instead of simply one enzyme, cellulase from most fungi is actually a collection of several distinct enzymes that work synergistically to accomplish the overall task of cellulose degradation. We can classify cellulase-secreting fungi into different groups: brown rots, white rots, and red rots. Unlike white and red rots that degrade cellulose and lignin, brown rots degrade mainly cellulose (Kirk 1976).

Several microorganisms are known to produce cellulase that hydrolyzes cellulose and hemicellulose. The best known is the filamentous fungi T. reesei. This microorganism generates at least three well-known enzymes known as: (a) b-1,4-d-glucan glucanohydrolase (an endo-enzyme),

(b) b-1-4-d-glucan cellobiohydrolase (an exo-enzyme), and

(c) b-glucosidase (Jeffries 1987). The most common cellulase, b-1,4-d-glucan glucanohydrolase or Cx, has several constituents. One of these constituents acts as the initiator of cellulose hydrolysis. The second enzyme, b-1,4-d glucan cellobiohydrolase, has two constituents: b-1,4-glucan gluco-hydrolase excises one glucose molecule from the nonreducing end of the cellulose chain, and b-1,4-glucan cellulobio-hydrolase removes two glucose units at a time from the end of the cellulose chain. It is further divided into two subcomponents CBHI and CBHII. The third class of enzyme, b-glucosidase, does not act directly on cellulose. It is nonetheless important because it hydrolyzes the glucose dimer cellobiose, which yeast cannot ferment, to liberate two glucose monomers, which yeast can readily utilize. Furthermore, cellobiose inhibits cellulase enzymes much more strongly than glucose does. The presence of sufficient quantities of b-glucosidase helps reduce feedback product inhibition.

Through pyrolysis, biomass is gasified into synthesis gas. Above 300°C, cellulose is degraded into volatile, gaseous products such as hydrogen, carbon monoxide, and carbon dioxide. At intermediate temperatures, decomposition is

Table 4 Yield of sugars from enzyme hydrolysis (5 wt% suspension) of acid and base treated solid based on 100 lb of original material

Conversion of available carbohydrate

Table 4 Yield of sugars from enzyme hydrolysis (5 wt% suspension) of acid and base treated solid based on 100 lb of original material

Conversion of available carbohydrate



Poly glucose



G & PG











Corn stover








Cotton gin trash








Rice hulls








Rice straw








Sorghum straw








Wheat straw








rather slow and the products formed are less volatile. Atmospheric conditions greatly affect pyrolysis. In the presence of oxygen, dehydrogenation and depolymerization occur quickly. On the other hand, in the presence of inert substances, depolymerization is slow, and unwanted byproducts appear (Wilke et al. 1983). In subsequently steps, the synthesis gas is bubbled into a submerged culture, and anaerobic microorganisms (e.g., bacteria Clostridium ljungdahlii) convert the pyrolysis product into ethanol (Klason et al. 1990). The chief obstacle of this technology is the high cost of energy (mainly electricity) to heat the biomass, and the low value of the synthesis gas thus produced. Pyrolysis treatment may be economically viable if the fermentation product is valuable.

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