708 Journal of Chemical Education _ Vol. 87 No. 7 July 2010 _ pubs.acs.org/jchemeduc _ r2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100260g Published on Web 04/28/2010 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 *jepstein1@spc.edu 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 pro- mise 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 pretreat- ment. 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). 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 (KH 2 PO 4 , 1.0 g/L; CaCl 2 , 0.10 g/L; MgSO 4 , 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 con- centration 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 CO 2 (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 Scheme 1. Conversion of Glucose to Ethanol