Objectives

Introduction

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 -------------> Glucose

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.

List of Reagents and Instruments

A. Equipment

• Erlenmeyer flasks
• Graduated cylinder
• Pipets, 1ml, 10ml
• Test tubes
• Incubator or thermostated shaker
• Temperature bath (or heat source -- Bunsen burner or hot plate)
• Thermometer
• Balance
• Syringe
• Filter holder
• Filter paper
• Spectrophotometer

B. Reagents

• Cellulose source (filter paper, wood chips, carboxymethyl cellulose, cotton)
• Cellulase, buffered at pH=5.00±0.01, 10g/l solution
• HCl, 5% solution
• H₂SO₄, 5% solution
• KOH
• Reagents for sugar analysis

Procedures

1. Enzymatic Hydrolysis: Repeat the same procedures for shredded wood chips (a complex and impure mixture of cellulose, lignin, and a variety of others), carboxymethyl cellulose (a model amorphours-structured cellulose), and cotton (90 % cellulose, mostly crystalline-structured). If time permits and if there is extra enzyme solution, try other sources of biomass and waste materials such as newsprint, grass, straw, and corn stalk. See Note 1.
   • Shred a 10 cm² piece of cellulose filter paper and weigh 0.1 g. (As opposed to other type of papers with binding materials, a piece of cellulose filter paper without wetting agents has minimum impurities and is almost pure in cellulose. The result of a quantitative analysis using a filter paper would have been very unreliable had impurities leached out into the filtrate.
   • Submerge the shredded paper in 10 ml of the buffered cellulase solution in a test tube. Note the starting time.
   • Incubate the mixture at 40°C. (The enzyme is most active at a temperature of 40°C and a pH of approx. 4.5.)
   • This reaction should last for approximately 24 hours. Take 1 ml samples at some predetermined appropriate intervals. Note that one does not have much to waste because the starting sample is small. (A volume of 1 ml is actually considered as a huge sample when working with biochemicals.)
   • Stop the hydrolysis reaction in the sample. The first method of stopping the reaction is to deprive the mixture of substrate. This can be easily achieved by filtering out the residual solid material from the solution. The individual samples may be stored frozen for later analysis. The samples are thawed and brought to room temperature before they are subjected to measurements. However, this first method is not applicable to soluble cellulose, e.g., CMC. Alternatively, the enzymatically catalyzed reactions can be halted either by adding a strong enzyme inhibitor or by raising the temperature of the mixture to 90°C for 5-10 minutes in a heated bath to inactivate the enzyme.
   • Measure the glucose concentrations of the samples with the dinitrosalicylate colorimetric method. (Reference: Gail Lorenz Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Analytical Chemistry, 31, 427, 1959.) See Note 2.
2. Acid Hydrolysis (Sulfuric Acid): Use the same cellulose sources as in enzymatic hydrolysis.
   • Add 0.2g cellulose to 10ml of 5% H₂SO₄ solution in a lightly capped test tube. See Note 3. One may choose to carry out the reaction at 90°C instead of at room temperature.
   • This reaction should last for 2 hours. Take 1 ml samples at some predetermined appropriate intervals.
   • Stop the hydrolysis reaction in the sample by neutralizing the acid and slightly reversing the pH with the addition of a small volume of a concentrated potassium hydroxide solution. Make a quick calculation to see how much KOH is needed for this purpose. Note that one has to keep a close track of the volume of KOH solution added because this information will be needed to calculate the glucose concentration in the original undiluted sample.
   • Measure the glucose concentration of the alkaline sample.
3. Acid Hydrolysis (Hydrochloric Acid): Substitute 5% sulfuric acid with 5% hydrochloric acid and repeat the same procedures as in sulfuric acid.

Discussions

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.

Notes

1. As in any other experiments, remember to include simultaneously a control experiment or a blank solution. An enzyme solution without any solid substrate can be used as one of the controls which will yield the level of glucose entrainment, if any, originally present in the enzyme preparation. This is especially important when one is unsure of the content of a complex solution. Furthermore, the result of enzymatic actions should be compared to another control experiment in which only water is added to the solid substrate. This second control will give the background leaching out of glucose from the substrate, if there is any at all. One should always guard against these possibilities.
2. One needs to outline how to measure the glucose concentration with the dinitrosalicylate colorimetric method. The extent of disclosure of the procedures associated with a particular analytical method in science communication is commonly comparable to, if not less than, what is said in this manual. It is critical that the student learns how to read scientific literature and obtain the necessary information from it.
3. Concentrated hydrochloric and sulfuric acids are among the most corrosive, dangerous chemicals. They are more so when heated. We all know how a splash of these acids can ruin one's clothing (cotton) and permanently and severely disfigure one's face. In an actual process, much more concentrated acids are used at even higher temperatures (180°C). Be sure to wear a pair of safety glasses to protect your eyes. Put on a lab coat or an apron to protect your expensive clothing. Thoroughly wipe up any chemical spill immediately before someone else puts his elbow over it.
4. Other assay methods may be used to measure the reducing sugar concentration. When the substrate is soluble, viscosity measurements in lieu of sugar measurements may be used to indicate enzyme activities. When the substrate is insoluble, turbidity and weight loss are sometimes used as indicators of enzyme activities.
5. The solution of commercially available cellulase can easily support the growth of molds and cannot be kept long at room temperature.

Questions

1. Based on sound engineering economics principles, you, as an engineer, now must make the choice between the two hydrolysis methods studied in this experiment. Justify your choice, preferably backed by a rough estimate for the unit cost associated with the glucose production. Compare this to the current market price of a related compound, say, sucrose (table sugar) or ethanol. (Make sure that your comparison is fair.) Is the proposed process profitable? What items or processing steps contribute significantly to the final cost? How can these costs be drastically reduced to make the process more attractive?
2. If you had the time and resources, what other experiments would you perform to reach better conclusions on the merits of acid hydrolysis versus an enzymatic one? %T and pH effects, percent acid used, source of enzyme, etc.
3. Is it possible to introduce a suitable bacterial flora into our intestinal tract so that we can digest grass as cattle do? If yes, why has it not been done to solve the problem of food shortage in some of the developing countries? If no, what makes it possible for cattle but not for humans?
4. With the help of better screening techniques and recombinant DNA technology, many scientists are actively engaged in the isolation/creation of a super bug that can digest lignocellulose at an extremely high rate. What are the potential damages if this organism is released to the outside environment? What safety features can a scientist deploy to minimize the impact of such an event that is certainly unavoidable if the organism is put to use in a large scale process?
5. Comment on ways to improve the experiment.

References

1. Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, 2nd Ed., p163-172, McGraw-Hill, 1986.
2. Bertran, M.S. and Dale, B.E., Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose, Biotech. Bioeng., 27, 177, 1985.
3. Linko, M., An evaluation of enzymatic hydrolysis of cellulosic materials, in Advances in Biochemical Engineering, 5, 39, 1977.
4. Ghose, T.K., Cellulase biosynthesis and hydrolysis of cellulosic substances, in Advances in Biochemical Engineering, 6, 25, 1977.
5. Grethlein, H.E., Comparison of the economics of acid and enzymatic hydrolysis of newsprint, Biotech. Bioeng., 20, 503, 1978. Erickson, L.E., Energetic efficiency of biomass and product formation, Biotech. Bioeng., 21, 725, 1979.