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
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with ethanol produced from sugar cane. In the United States,
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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).
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708 Journal of Chemical Education 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
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 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 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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 In the Laboratory

Table 1. Production of Ethanol from One Student

carbohydrate ethanola vol of ethanol per
source (%) 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
a
Ratio of volume of ethanol recovered during distillation to the total volume
distilled.

saddles (18). The ethanol content of each fraction is determined

Figure 1. Typical student results of ethanol yield expressed as volume of
by density.
ethanol produced per hectare of land in one year (1 ha is 2.47 acres).

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 com-
pounds can be slightly irritating to the skin and eyes: KH2PO4,
MgSO4, NaCl, CaCl2, ammonium tartrate, cellulase, and ampi-
cillin. 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 produc-
tion 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 fermenta-
tion. 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
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 techno-
logy. 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), demon-
strating 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 experi-
ment 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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2007, 31, 416–425.
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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.
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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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710 Journal of Chemical Education Vol. 87 No. 7 July 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc.