The Sustainable Development of Ethanol for Environmentally Friendly Alternative Energy

The Problem

The quest for alternative fuels began with the realization that the oil supply was finite. Since 1975, researchers have looked for economical and nationally viable ways to produce alternative energy, preferably from abundantly available, biodegradable, and renewable raw materials. Although much progress has been made, significant progress towards a viable source is still in infancy. Ethanol is an excellent transportation fuel, in some respects superior to gasoline. In comparison to gasoline, neat ethanol burns more cleanly, has a higher octane rating, can be burned with greater efficiency, is thought to produce smaller amounts of ozone precursors (thus decreasing urban air pollution), and is particularly beneficial with respect to low net Carbon Dioxide put into the atmosphere. Ethanol is considerably less toxic to humans than is gasoline (or methanol). Ethanol also reduces smog formation because of low volatility; its photochemical reactivity and that of its combustion products are low, and only low levels of smog-producing compounds are formed by its combustion. Its high heat of vaporization, high octane rating, and low flame temperature yield good engine performance.Currently 95% of ethanol being produced globally is by the use of grain and only 5% is through the cellulosic source. However, the potential quantity of ethanol that could be produced from cellulose is over an order of magnitude larger than the current production from corn. In contrast to the corn-to-ethanol conversion, the cellulose-to-ethanol route involves little or no contribution to the greenhouse effect and has a five times better net energy balance. Along with this, a survey of US research teams showed the ratio of total energy contained in a liter of ethanol to the nonrenewable energy consumed during production of a liter of ethanol, revealed that grain-ethanol has an average return of 138% and cellulosic ethanol has an average return of 363%. As a result of such considerations, microorganisms that metabolize cellulose have gained prominence in recent years. Currently, use of enzymes has driven the cost of the cellulose-to-ethanol process up, thus a great deal of effort has gone into the process of converting cellulose to ethanol directly via a cheap and efficient process. Therefore, lately a large amount of focus has dealt with Clostridium thermocellum, a thermophilic (grows at 60ºC), anaerobic, and cellulolytic bacterium. C. thermocellum has the ability to convert cellulose to ethanol is an efficient one step process by combining the enzyme production, hydrolysis and fermentation steps of the process. Besides the potential key to solving the global energy crisis, focus on this bacterium has been because of its many advantages. First, C. thermocellum can utilize lignocellulosic waste and generate ethanol, a rare property among living organisms. Also, since the optimum temperature for the cultivation of the organism is 60°C, the problems of contamination are lessened and the cooling of large fermentors is much simplified. Finally, growth at a high temperature facilitates the recovery of ethanol. A process designed for ethanol production from cellulose had a selling price of U.S. $1.45 in 2006, whereas production from corn had a price of $4.69 per gallon. Thus, substantial cost reductions are possible if C. thermocellum is used, and could make biological ethanol a competitive fuel. With the investigations reported here, ethanol can perhaps be produced for even less. Degradation products such as acetate and lactate that decrease the yield of ethanol and can act as weak uncouplers and slow cell growth is the primary hurdle with using the bacterium. When using complete bacterium, C. thermocellum, the final yield of ethanol is limited. Regulation of cellulase synthesis by C. thermocellum is central determinant of hydrolysis and growth rates and thus is of interest for understanding cellulose utilization in both natural environment and in industrial processes. Recently, a new gene cluster of 5 genes of C. thermocellum was identified. It is the largest cellulosomal gene cluster in the microorganism. The investigations reported here focused on this newly found gene cluster. Separating individual genes without the use of denaturants or limited proteolysis has had only partial success. In the investigations reported here, molecular cloning and protein expression were used to circumvent this.

Plan of Action

METHODOLOGY 1) Organism The C. thermocellum strain used was NCIB 10682, also indexed as ATCC 27405. 2) Gene Identification of Gene 2 and 5 To begin the process of cloning, Sequence Homology, or the comparison of our gene of unknown function to sequences of known functions is done using a BLAST (Basic Local Alignment Search Tool). BLAST compared the unknown sequences to sequences of known functions. These genes were compared to all the genes in the GenBank for homology. Gene 2 and 5, of a new gene cluster, were tested because significant homology existed for these two genes with various cellulases. 3) Molecular cloning of Gene 2 and Gene 5 Amplification and molecular cloning of Genes 2 and 5 was initiated with Polymerase Chain Reaction (PCR) in a four step process. These products were purified using GFX columns with Tris-HCl buffer, and verified on agarose gel electrophoreses. Using restriction enzymes XhoI and EcorV, the genes, along with plasmid pTBX1, were cut to generate sticky ends. Ligation joined them together into cohesive products, which were then transformed into Escherichia coli for the final clone. Mini-prep techniques isolated the plasmids and purified the products, which were verified with another group of agarose gel electrophoreses. Protein expression was induced by IPTG for both genes, and was purified via chitin binding column and heat purification. Final protein expression was verified on a SDS polyacrylamide gel electrophoreses. 4) Cellulose Degradation: Reducing Sugars Assay at 540 nm This assay is used to calculate the amount of reducing sugars the protein can produce upon cellulose hydrolysis. The cellulosic materials used for this assay were as follows: Avicel, CMC, Lichenan, Chitin, Xylan from oat-spelt and Xylan from birch wood. 5) Cellulose Hydrolysis: Turbidity Absorbance Assay at 660 nm This assay measures the ability of the protein of Gene 5 or Gene 2 to degrade water insoluble cellulosic material to soluble sugars. The absorbance at 660nm determines how much of the cellulosic material is broken down. The cellulase enzyme used is the expressed protein. The cellulosic materials were Xylan from birch wood and Avicel (pure cellulose). This test will only be used if there is an indication of cellulose degradation via the Reducing Sugars Assay. The assay was also used to study various combinations of C. thermocellum and Gene 5. A standard curve was also created with C. thermocellum alone and with Gene 5. C. thermocellum produces an extracellular cellulase system capable of degrading crystalline cellulose (3). Studies of the C. thermocellum cellulase system are important due to the potential use of this bacterium in the direct conversion of cellulosic’s to ethanol. Genes 2 and 5 belongs to a novel gene family of C. thermocellum. There is no current published literature concerning Genes 2 and 5 of C. thermocellum. After successfully cloning and amplifying the genes using PCR, the DNA and plasmid pTBX1 were cut with restriction enzymes and ligated together. The transformation of the ligated product into the bacterial host cell (E. coli) was successful based on the results of Agarose gel Electrophoresis. Finally expression of the recombinant protein was successful based on the Sodium-lauryl Sulfate- (SDS-) Polyacrylamide Gel results. The activities of the recombinant proteins were tested on various cellulosic’s in the Reducing Sugars and Turbidity Absorbance assay. Gene 2 was unable to degrade cellulose significantly. Thus, with Gene 2, cellulose degradation is problematic and unlikely to offer benefits in the future. The investigations reported here showed that the Gene 5 product could break down Avicel, a pure form of cellulose very efficiently. Other cellulosic materials (Xylan from oat-spelt, Xylan from birch wood, carboxymethylcellulose (CMC), chitin, and lichenan) also were hydrolyzed by the Gene 5 product to reducing sugars at efficiency of 20-40% of Avicel. Gene 5 is an abundant subunit within its cellulosome and so far is the one of the only subunits that has been demonstrated to degrade crystalline cellulose. Furthermore, Gene 5 has been classified as an exo-enzyme. An exo-enzyme cannot generate multitudes of reducing sugars from cellulosic materials like carboxymethycellulose (CMC), modified cellulose, i.e. carboxymethyl group are attached to glucose moieties. Since an exo-enzyme "chews" from an end of the CMC chain, its action is blocked when it hits any carboxymethylated glucose moiety, generating low activity on CMC. However, the cellulose chain of Avicel is not carboxymethylated and an exo-enzyme can continue to "chew" without being blocked to produce glucose or cellobiose into the solution, generating high activity on Avicel. The exo-enzymatic activity of Gene 5 presented in this work is consistent with the generally accepted concept that the exo-enzyme is crucial for degrading crystalline cellulose efficiently, and the rarity of finding one. The investigations involving the combination of C. thermocellum and excess Gene 5 had novel findings. When any amount of C. thermocellum was compared to C. thermocellum in combination with Gene 5, the sample with Gene 5 consistently degraded crystalline cellulose faster and with higher efficiency. Furthermore, Gene 5 was tested on crystalline cellulose alone. At various concentrations tested, the amount of Gene 5 degraded crystalline cellulose more efficiently than C. thermocellum. Protein concentrations were equal with the C. thermocellum protein and Gene 5. Thus it was concluded that Gene 5 can degrade crystalline cellulose efficiently, and that in the future, it could be used to efficiently produce ethanol. Gene 2 was not very effective in degrading cellulosic materials. However, Gene 5 has a significant role in C. thermocellum’s bacterium in the ethanol production process from cellulose, and can be used in the future for efficient ethanol production. CONCLUSIONS The findings of these investigations are novel and significant. • The discovery of Gene 5 as an exo-enzyme, an extreme rarity. Very few exo-enzymes have been found before, and these investigations have successfully identified one more. Exo-enzymes are able to degrade cellulose very efficiently. • The fact that Gene 5 aids in ethanol production, and with increased amounts of Gene 5, increased biomass degradation is observed. • Novel discovery of Gene 5 could be the key to creating ethanol efficiently and cheaply for sustainable development from biomass in the future.

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