There are two types of hydrogen bonds in cellulose molecules: those that form between the C3 OH group and the oxygen in the pyranose ring within the same molecule and those that form between the C6 OH group of one molecule and the oxygen of the glucosidic bond of another molecule. Ordinarily, the beta-1,4 glycosidic bonds themselves are not too difficult to break. However, because of these hydrogen bonds, cellulose can form very tightly packed crystallites. These crystals are sometimes so tight that neither water nor enzyme can penetrate them; only exogluconase, a subgroup of cellulase that attacks the terminal glucosidic bond, is effective in degrading it. The inability of water to penetrate cellulose also explains why crystalline cellulose is insoluble. On the other hand, amorphous cellulose allows the penetration of endogluconase, another subgroup of cellulase that catalyzes the hydrolysis of internal bonds. The natural consequence of this difference in the crystalline structure is that the hydrolysis rate is much faster for amorphous cellulose than crystalline cellulose. The process of breaking the glucosidic bonds that hold the glucose basic units together to form a large cellulose molecule is called hydrolysis because a water molecule must be supplied to render each broken bond inactive. In addition to crystallinity, the chemical compounds surrounding the cellulose in plants, e.g. lignin, also limit the diffusion of the enzyme into the reaction sites and play an important role in determining the rate of hydrolysis. Sometimes, wood chips are pretreated with acid at approximately 160°C to strip hemicellulose and lignin before they are treated with an enzyme or a mixture of enzymes. In general, 20 to 70% yield of glucose can be expected after 24 hours.
The conversion of cellulose into glucose is now known to consist of two steps in the enzyme system of Trichoderma viride. In the first step, beta-1,4 glucanase breaks the glucosidic linkage to cellobiose, which is a glucose dimer with a beta-1,4 bond as opposed to maltose, a counterpart with an alpha-1,4 bond. Subsequently, this beta-1,4 glucosidic linkage is broken by beta-glucosidase:
b-1,4 glucanase b-glucosidase Cellulose ---------------> Cellobiose -------------> GlucoseThe kinetics of cellulose hydrolysis has been widely studied, and Michaelis-Menten types of rate expressions with substrate or product inhibition terms have been proposed to describe the observed reaction kinetics.
A wide variety of fungal and bacterial species produce cellulase and transport the enzyme across the cell membrane to the outside environment. Although it is common to refer to a mixture of compounds that can degrade cellulose as cellulase, it is really composed of more than one distinctive enzymes. Recent research has shown that one of the components is relatively inert with the ability of recognizing and attaching itself to the surface of the cellulose mass, in addition to the ability of recognizing and holding onto another protein component that exhibits enzymatic activities. Thus, the chance of reaction is significantly enhanced by a proximity effect, because the active enzyme is held onto the surface of a solid substrate by an inert protein which acts as a glue.
The species most often used to study the production of cellulase are white-rot fungal cultures of Trichoderma ressei and Trichoderma viride. We all have seen a piece of rotting wood. And perhaps without knowing it, we are actually quite accustomed to the appearance and action of this fungi. As in Experiment No. 1, it is only natural that the most promising place to search for cellulase is in a piece of rotting wood. The microorganisms responsible for this enzyme can easily be isolated from a piece of rotting wood, or from a termite's gut if bacterial species are desired. Other fungal species often used are Fusarium solani, Aspergillus niger, Penicillium funicolsum, and Cellulomonas sp. The bacterial species Clostridium thermocellum and Clostridium thermosaccharolyticum also represent promising candidates for cellulase production because they are thermophilic (less contamination problem and faster rate at a high temperature), anaerobic (no oxygen transfer limitation), and ethanologenic (conversion of cellulose to ethanol via glucose with a single culture). In general, different species of microorganisms produce different cellulolytic enzymes.
There has been a large amount of research work done on the digestion of cellulose into glucose. The generated glucose can be used to produce single cell protein as food for livestock or even for humans. Glucose can also be used as the starting raw material in the production of a wide variety of chemicals and fuels. This is usually carried out with the help of microorganisms. For example, glucose can be easily fermented to ethanol by Saccharomyces cerevisiae (yeast) or Pseudomonas mobilis (bacterium). Ethanol can be used as gasoline or processed further to make other common petrochemicals. Another example is the conversion of glucose into solvents such as acetone and butanol by Clostridium acetobutylicum. Because the volume of cellulose is so overwhelming and because the resource is renewable, the world will likely to depend on it more heavily for food, fuel, chemical supplies, and raw materials in the future. It has the great potential of alleviating the need for petroleum, whose supply is fast dwindling.
Thus, the ability to manipulate this organic chemical has extremely important implications. A breakthrough in the investigation of cellulose digestion processes will not only have an enormous impact on the world food supply, economy, and geopolitical balance of power, it will also greatly influence the various types and ways products are produced by the chemical industry and enjoyed by the end users. This experiment introduces a student in biochemical engineering to one of tomorrow's technologies with the most far-reaching impacts.
As demonstrated in this experiment, the breaking down some of the cellulose is really not very difficult. However, translating a process from a laboratory scale to a commercial scale is not so trivial. First of all, the entire operation has to be both technically sound and economically feasible. In order for a process to be actually adapted, it, of course, has to be technically possible first. In addition, it must offer some clear advantage over all other competing processes. This advantage is almost always measured in the form of a larger profit margin, irrespective of the political system in which the process is to be employed. Note that in calculating the profit, one must duly include various costs that are sometimes not obvious nor easy to estimate, e.g. the public images, institutional responsibilities, and environmental impacts. Unprofitable processes are a waste of natural and human resources and must not survive. As a chemical engineers, whether conducting basic research or designing a plant, one is continually reminded of the economical impact.
Two typical approaches to effect a similar end result are studied in this experiment. However, one should keep in mind that there are numerous other competing approaches, and one is constantly faced with multiple choices. For example, acetic acid can be produced by fermentation means or chemical synthesis. So are a wide range of pharmaceuticals. As a matter of fact, life is rarely simple and straight forward enough that there is only one choice.