In the Laboratory
Developing Biofuel in the Teaching Laboratory: Ethanol from Various Sources Jessica L. Epstein,* Matthew Vieira, Binod Aryal, Nicolas Vera, and Melissa Solis Department of Chemistry, Saint Peters College, Jersey City, New Jersey 07306 *
[email protected] Ethanol fuel is experiencing a period of growth. In the United States, ethanol is currently used in gasoline blends (1, 2). Brazil provides a successful model for proponents of ethanol fuel (1, 3): after thirty years, Brazil has nearly replaced fossil fuel with ethanol produced from sugar cane. In the United States, where sugar cane crops are impractical, corn is one of the primary carbohydrate sources for ethanol. A typical ethanol plant (4) uses fermentation (Scheme 1) to produce ethanol from domestically grown corn. Ethanol is then separated from the fermentation mixture by fractional distillation. Objections to corn-based ethanol are both ethical and practical (5). Corn crops for fuel divert land away from food crops, produce less sugar than sugar cane, and have a significant material cost and energy input including the fuel for plowing, seeding, irrigation, harvesting, and application of fertilizers and pesticides (6, 7). Perennial, cellulosic sources such as switchgrass offer promise because large quantities of grass are harvested with no energy input for upkeep (8). Waste biomass from food crops, such as wheat straw and corn stalks, are also promising because there is almost no additional cost or energy input (9). Unfortunately, these cellulosic sources present two problems. First, the glucose (the fermentable carbohydrate) exists as cellulose, which can be difficult to break down into simple sugars (10). The second problem is lignin, a structural component of plants (11). Lignin serves as a binder for cellulose fibers in plants and adds strength and stiffness to the cell walls. Lignin is a large, hydrophobic molecule that does not easily separate from cellulose. For this reason, many research laboratories focus on switchgrass, which does not contain lignin (12). This laboratory exercise explores the production of ethanol from fruits, grains, and grass, using different techniques to free the glucose from the starch or cellulose form. Whereas previous experiments explored ethanol production from corn (13, 14) and more recently newspaper (15), we explore production from new carbohydrate sources. The data from several carbohydrate sources are then combined to demonstrate the potential of each source for large-scale ethanol production. Fermentation Procedure Fruit Juice Apple or grape juice is fermented directly with no pretreatment. Fruit juice, 200 mL, is placed in a 500 mL round-bottom flask with 3.0 g of dry yeast (Fleishman's, dry active). No additional nutrients or changes in pH are needed to grow the yeast cells. The mixture is swirled to dissolve the yeast. The preparation time is 30 min. The fermentation is then carried out (see below). 708
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Scheme 1. Conversion of Glucose to Ethanol
Corn or Potato Starch Frozen corn, 100 g, is pureed with 50 mL of water, or potato starch, 50 g, is mixed with 100 mL of water. Concentrated acid solution, 25 mL of 6.0 M HCL, is added, and the mixture is heated to 90 °C for 45 min. Glucose from the starch is liberated by the acid treatment. The mixture is neutralized with 25 mL of 6.0 M NaOH, and the pH is adjusted to 6.2 with phosphate buffer. The glucose content is assessed with a glucose test strip (Carolina Laboratories) and should be ∼0.1 M. Yeast, 3.0 g, is added to the treated corn mash, or yeast, 3.0 g, and minimal medium (KH2PO4, 1.0 g/L; CaCl2, 0.10 g/L; MgSO4, 0.5 g/L; ammonium tartrate, 10 g/L; and NaCl, 0.1 g/L) are added to the treated potato starch. The preparation time is 75 min. The fermentation is then carried out (see below). Grass Dried, ground grass, 2.0 g, is pretreated by heating to 90 °C with 2% NaOH for 90 min and then is washed with hot water (16). Cellulose is freed from other cellular components by the alkaline treatment. Excess water is removed by vacuum filtration, and the treated grass is stored at 4 °C until the day of the experiment. The treated grass is incubated with cellulase enzyme (30,000 units), 0.1 M acetate buffer at pH 5.0, and 0.1% ampicillin (to prevent bacterial growth) for 24 h. Glucose from cellulose is selectively hydrolyzed by cellulase. The glucose concentration is measured, and the pH is adjusted with phosphate buffer (pH 6.2, 0.1 M). The yeast and minimal medium (see above) are added. The preparation time is 2.5 h with a 24 h incubation period. The fermentation is then carried out. Fermentation The fermentation mixture is placed in a round-bottom flask fitted with a one-hole rubber stopper connected to a piece of bent glass tubing. The other end of the glass tubing is attached to Teflon tubing and is submerged below the surface of a saturated solution of aqueous calcium hydroxide to exclude atmospheric oxygen and to absorb CO2 (17, 18). In all cases fermentation requires one week. Fractional Distillation The fractional distillation is performed using standard organic chemistry glassware and a column packed with ceramic
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Vol. 87 No. 7 July 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100260g Published on Web 04/28/2010
In the Laboratory Table 1. Production of Ethanol from One Student ethanola (%)
vol of ethanol per mass of biomass (mL/g)
apple juice
5.7
0.060
grape juice
7.2
0.070
corn
6.2
0.14
potato starch
6.0
0.27
grass
1.2
0.33
carbohydrate source
a
Ratio of volume of ethanol recovered during distillation to the total volume distilled.
saddles (18). The ethanol content of each fraction is determined by density. Hazards Concentrated HCl and NaOH are highly corrosive. The ethanol product is flammable. Celite powder is an eye and respiratory irritant. In the powder form, the following compounds can be slightly irritating to the skin and eyes: KH2PO4, MgSO4, NaCl, CaCl2, ammonium tartrate, cellulase, and ampicillin. CaCl2 is also a respiratory irritant. Yeast, cellulase, ampicillin, and the fermentation products of this experiment may be hazardous in the case of ingestion or inhalation (ethanol is a central nervous system depressant). Heating mantles, hot plates, and glassware can become very hot. Eye protection and laboratory coats should be worn at all times. Detailed safety information is provided in the supporting information. Results and Discussion We introduce these experiments during the first semester of an organic chemistry course, when students learn fractional distillation and filtration as part of a series of experiments designed to build laboratory skills. Discovering the power of chemistry to tackle politically and socially relevant issues such as renewable energy enhances student learning. Other instructors may want to introduce this experiment in a general chemistry or environmental science course, where students only find the percent ethanol from the density of their filtered fermentation broth and omit the fractional distillation. We divide the students in each laboratory section into two groups, with each group producing ethanol from a different carbohydrate source. For example, students in a laboratory section can compare ethanol production from juice versus starch or starch versus cellulose. To avoid consuming two laboratory periods, the fermentation is set up a week ahead at the end of a regularly scheduled laboratory. Apple juice fermentation produces 4.6-5.7% ethanol, whereas grape juice produces 6.8-7.3%. Typical data from one student is shown in Table 1. The difference in ethanol production can be explained by comparing the sugar content listed on the juice bottles (apple juice, 510 mM; and grape juice, 625 mM). Both the corn and potato starch fermentations produce 4.0-6.2% ethanol. Prior to fermentation, it is important for students to check sugar content before they proceed to fermentation. Theoretically, fermentation can produce up to a 12% ethanol solution. However, the corn mash is quite thick, so increasing the quantity of corn would prevent contact with the yeast and actually decrease the ethanol production. We chose not
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Figure 1. Typical student results of ethanol yield expressed as volume of ethanol produced per hectare of land in one year (1 ha is 2.47 acres).
to use cornmeal or grits because these forms of the grains sink to the bottom of the fermentation flask and require continuous mixing, which is another energy input. The grass fermentation produces 1.0-1.3% ethanol, and most students report glucose concentrations ranging from 100-150 mM. We provide the students with grass that is already pretreated to fit this experiment into the curriculum. The preparation of the grass for fermentation highlights some of the challenges facing this new technology. The addition of enzyme and ampicillin (to prevent bacterial growth) has not been tested on an industrial scale, which is why many laboratories have cloned the cellulase gene directly into yeast (10). Students initially find percent ethanol, but this approach does not fully evaluate the effectiveness of a carbohydrate source. Percent ethanol is highest for the grape juice, and students often conclude that fruit juice is the most efficient followed by starch and finally cellulose (grass). Although percent ethanol is higher for the fruit juices, the starch and cellulose sources produce more ethanol per gram of biomass than either juice (Table 1), demonstrating that typical student results agree with the current analysis of ethanol fuel (8, 9). Grass produces the most ethanol per year on a hectare of land (Figure 1) because perennial grasses grow quickly and are harvested several times per year. Summary In this experiment, routine laboratory techniques can be infused with relevance, making them more meaningful to the students. Most students are familiar with fermentation and distillation in the production of alcoholic beverages, but few understand the applications of these techniques in petroleum processing and the development of alternative fuel. The experiment can be limited to techniques only, or students can be asked to research the subject further in their reports, where they discover that cellulosic ethanol sources offer great promise but the technology is still developing. Literature Cited
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1. Solomon, B. D.; Carnes, J. R.; Halvorsen, K. E. Biomass Bioenergy 2007, 31, 416–425. 2. Barry, P. Science News 2007, 173, 49. 3. Bourne, J. K. Biofuels: Green Dreams. Natl. Geogr. 2007 (October). 4. E3 Biofuels. http://www.e3biofuels.com/ (accessed April 2010). 5. Johnson, J. Chem. Eng. News 2007, 85 (1), 19–21. 6. Hill, J.; Nelson, E.; Tilman, D.; Polasky, S.; Tiffany, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 102, 11206–11210.
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In the Laboratory 7. Pietro, W. J. J. Chem. Educ. 2009, 86, 579–581. 8. Schmer, M. R.; Vogel, K. P.; Mitchell, R. B.; Perrin, R. K. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 464–469. 9. Ritter, S. K. Chem. Eng. News 2004, 82 (22), 31–34. 10. Demain, A. L.; Newcomb, M.; Wu, J. H. Microbiol. Mol. Biol. Rev. 2005, 69, 124–154. 11. Albersheim, P. E. Sci. Am. 1975, 234, 80–95. 12. National Renewable Energy Lab. http://www.nrel.gov/technologytransfer/ (accessed April 2010). 13. Oliver, W. R.; Kempton, R. J.; Conner, H. A. J. Chem. Educ. 1982, 59, 49–52. 14. Maslowsky, E. J. Chem. Educ. 1983, 60, 752. 15. Mascal, M.; Scown, R. J. Chem. Educ. 2008, 85, 546–548.
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16. Silverstein, R. A.; Chen, Y.; Sharma-Shivappa, R. R.; Boyette, M. D.; Osborne, J. Bioresour. Technol. 2007, 98, 3000–3011. 17. Mohrig, J. R.; Hammond, C. N.; Morrill, T. C.; Neckers, D. C. Experimental Organic Chemistry; W. H. Freeman and Co.: New York, 1999; pp 25-29. 18. Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Introduction to Organic Laboratory Techniques: A Small Scale Approach, 2nd ed.; Thompson Brooks/Cole: Belmont, CA, 2005; pp 132-134.
Supporting Information Available Student handout; information for the instructors. This material is available via the Internet at http://pubs.acs.org.
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