Cellulosic Ethanol: Energy for the Future!

Wendy Guo and Jenny Wang Background

One of the key challenges that the world faces today is to create a safe, efficient, and sustainable energy source to support our energy needs. On the topic of fuel, it is necessary to realize that gas is not only expensive but also very damaging to the atmosphere. Biofuels have a great deal of potential as it is still a developing alternative fuel. Created from organic matter, it is sustainable due to the fact that plants absorb the same amount carbon in their lifetime as when they are used up as fuel. [1] Furthermore, the most promising form of biofuel is cellulosic ethanol, since it can be made from a variety of waste materials such as corn husk, sugarcane molasses, citrus peels, and as well as leaf litter. This is an advantage to common corn ethanol because rather than taking up land to grow this staple crop, organic wastes can be used instead. [2]

Figure 1. Cellulose and starch: structure and hydrolysis

There are two main helpers that are required for the decomposition of ethanol from cellulosic materials, which are enzymes and yeast. Cellulase is an enzyme that is used for hydrolysis of cellulose. [3] After the cellulose has been broken down, an enzyme called amylase is needed to catalyze the breakdown of starch into sugars, both depicted in Figure 1. [4] Once the sugar is produced, yeast is used to help produce alcohol as shown in the reaction in Figure 2. The specific strain of yeast used that optimizes the amount of alcohol produced is Saccharomyces cerevisiae, more commonly known as distillers yeast.
26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!

Figure 2. Fermentation of glucose by yeast.

Purpose
The purpose of this project is to improve part of the production process of cellulosic ethanol so that it is more efficient and cost effective. This project tests for the optimal amount of time required for fermentation by yeast to turn cellulosic materials into ethanol fuel.

Hypothesis
There were two hypotheses created for this experiment. The first one explains that the mixture of yeast, dextrose, and using enzymes would create the most yield: if the mixture of cellulose with enzymes, yeast, and dextrose creates the most ethanol than other mixtures of the above ingredients, then it is the most effective combination in producing cellulosic ethanol in a certain period of time. This was used to prove that this solution would be more effective over the other ones. For testing the optimal number of days required for fermentation, a question was posed due to the fact it was unclear as to how many days this could take. Also, many papers had a large range of the amount of optimal time, anywhere from half an hour to as much as two weeks.

Procedure
To begin, two master flasks were prepared. They are both composed of Metamucil fibre powder (cellulose) dissolved in distilled water. Flask A was treated with enzymes (cellulase and amylase), while flask B was not, being the control. Both also contained a buffer to maintain the pH level neutral. The master flasks were kept in a water bath (homemade incubator) for three days. This kept the enzymes working at its optimal temperature of 37 degrees Celsius. After 3 days, a total of 7 smaller flasks were created from the master flasks. The solution from flask A (treated with the enzymes) was added into flasks 1A, 3A, and 5A, while the solution from flask B was added to flasks 2B, 4B, as well as 6B. Flasks 1A and 2B had nothing
26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!

added. This was the positive control to show that no alcohol was being produced without the help of yeast. Next, flasks 3A and 4B had yeast added. Flasks 5A and 6B had yeast and 2% dextrose added. Dextrose was added so that if the yeast ran out of cellulose, then there would be supplementary sugar. There was one more flask, flask 7, was with no master solution but just distilled water, yeast, and 2% dextrose. This was the negative control to ensure that the yeast was working. Instead of measuring the amount of ethanol produced (which would require distillation), carbon dioxide production was recorded because in the decomposition reaction, ethanol and carbon dioxide are directly proportional to one another. The experiment was first tested by placing balloons onto the flasks to represent the carbon dioxide being produced and to collect data on the amount of gas. However, when weighing the flasks with the balloons, the data collected was inaccurate. This is because the more gas being produced within the flask, the more likely it is to effuse out due to the higher pressure. Therefore, trials from the balloons were used for visual representation. From this, eudiometers were used to collect accurate data as gas produced could be measure by water displacement. The first 10 trials were done by placing balloons on top of each flask to get a visual representation of the CO2. Eventually, 10 more trials were accomplished with eudiometers to obtain more accurate data of the carbon dioxide, and were once again checked daily to obtain the amount of gas being produced.

Results

Figure 3. CO2 Production Rate, Average of 10 trials SD

26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!

Flask 5A produced the largest volume of CO2 as expected, which consists of a mixture of cellulose treated with enzymes as well as yeast and 2% dextrose. Flask 3A follows shortly behind, which is a mixture of cellulose treated with enzymes and yeast. The other flasks did not produce nearly as much CO2, as seen in Figure 3. From this, it is evident that both enzymes (from master flask A) as well as yeast (placed in flasks 3 and 5) are essential for successful ethanol production. As shown in the graph, the slope of the first three days does not increase by much. However, during days four to six, the slope significantly increases. By day five or six, the line begins to plateau.

Figure 4. ANOVA Comparison of CO2 Production Next, the data was statistically analyzed using ANOVA (Analysis of Variance tests). The results were inputted in Excel, comparing each of the 7 flasks for each and every day. In Figure 4, this statistical test showed that the peak fermentation occurs before the end of day 5. The test also reveals that flask 5 is indeed the optimal mixture for ethanol production. Using yeast, enzymes (cellulase and amylase), and dextrose is the best combination for optimal efficiency.

Conclusion
In conclusion, if biorefineries were to utilize this information, using the solution of cellulose, distilled water, cellulase, amylase, yeast, and dextrose, then fermenting for five days would be the most cost efficient and rational choice. It was clear that all flasks other than 3A and 5A could be disregarded; only these two solutions should be put to use in the future.

26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!

Regarding the 5 days of fermentation time, this is beneficial for production as large batches can be allowed to ferment for a relatively short period of time. The addition of cellulase and amylase most definitely assisted in breaking down the cellulosic material for more convenient fermentation by yeast. This data is crucial for when applied in large scale production because budgets come into play. It is important to know when and how much yeast or enzymes to utilize to produce optimal amounts of ethanol, especially when used as a fuel. By using the information from our project, a great amount of costs can be reduced and in turn, more ethanol can be produced.

Further Studies
We would like to continue our research by running more trials to obtain more data and take eudiometer measurements past 7 days. Furthermore, we want to continue experimenting to better other parts of the production process, such as temperature, quantity and comparing different feedstocks. An organized database of optimized biofuel information is a goal towards which we are striving toward accomplishing, so that new research can be shared more easily between scientists and biofuel companies. We hope that this sustainable energy source will be soon utilized worldwide, with a set of optimized standards to make the process effective and cost-efficient. And weve worked hard to take a small step towards this huge goal.

Acknowledgements
Many people have enlightened us with their valuable opinions and/or encouraged us to pursue this project. We couldn't be more thankful to the following people: Catherine Anderson, Flora Ross, Pamela Simmonds, Yichen Zhang, Tiffany Lu, and last but not least, we would like to thank our parents for taking an interest in our project. We couldnt have done it without their support!

References
[1] IPIECA Biofuels Task Force, Biofuels, sustainability, and the petroleum industry (2009) [2] D. Bielle, Can Ethanol from Corn Be Made Sustainable? Scientific American: Science News, Articles and Information (2013)
26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!

[3] X. Jin, Breaking Down Cellulose, Stanford Phys. (2010) [4] N. S. Wang, Starch Hydrolysis by Amylase, Maryland Chem. & Biomol. Eng. [5] M. Knauf and K. Kraus, Specific yeasts developed for modern ethanol production, Ethanol Tech.

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26 April 2013 - Wendy Guo and Jenny Wang- Cellulosic Ethanol: Energy for the Future!