LaBelle—Miley

Introduction

It is common knowledge that petroleum oil is not a renewable resource

and will eventually be used up. However, what most people don’t realize is just

how fast the supplies of oil are being depleted. “In the time it takes most people

to read this sentence, the world will have used up (forever) about 8,000 barrels of

oil - 336,000 gallons; at 1000 barrels per second” (Some). Needless to say, it’s

going fast. Because of this, alternative and renewable fuel sources are

desperately needed. A biofuel is a fuel produced from renewable biomass

material, commonly used as an alternative fuel source. It is considered a carbon

neutral which means that it produces the same amount of carbon dioxide during

burning as during growth. This makes it a very safe and environmentally friendly

fuel source. More importantly, it is renewable. Ethanol is a type of biofuel made

directly from naturally grown plant matter. The most commonly used,

convenient, and cheapest (for the United States) ethanol source is

Saccharomyces cerevisiae. Due to this fact, the experiment performed was

designed to improve upon the use of corn as an ethanol source by changing the

temperature and amount of yeast during fermentation to produce the highest

alcohol content with the same amount of plant material. Alcohol content is

directly related to how well a material can be used as a biofuel. The higher the

alcohol content the better it is.

The purpose of this experiment was to determine what amount of

Saccharomyces Cerevisiae (yeast) and what temperature during fermentation

would yield the highest alcohol concentration in Zea maize (corn). In an attempt

LaBelle—Miley

to yield better and more efficient ways to produce ethanol the experimenters

used the data collected to determine which method of ethanol production and

which conditions yielded the highest alcohol content and therefore yielding the

best potential ethanol.

In this experiment, corn was mixed with yeast to be fermented, and then

the alcohol content was measured. First, corn was blended with water until it

was a paste-like mixture. Using a hydrometer, the alcohol content was

measured before the yeast was added. Depending on which trial, a certain

amount of yeast was added to the mixture. The mixture was then poured into a

two liter bottle and a balloon was placed over the mouth of the bottle. According

to the trial number, the bottle was placed in an incubator set at 22, 25, or 28 °C.

After four days, the bottle was removed from the incubator. The mixture was

strained and the alcohol content after fermentation was taken. This number was

subtracted from the first alcohol content to give the final alcohol content.

The practical application for this research was to find a way to get a higher

alcohol content (after fermentation) with the same amount of corn. By doing this,

it makes the production of ethanol more efficient for the companies. If a

company can use the same amount of corn to produce more alcohol it can save

them money, time, and resources. The higher alcohol content makes the ethanol

a better gas product for cars. If the companies could find a way to produce a

more efficient ethanol product, they could replace the depleting fossil fuel supply

with ethanol and not have to worry about eventually running out.

LaBelle—Miley

Review of Literature

The imminent shortage of gasoline is causing a large amount of research

on alternative fuel manufacturing, selling, and creation of cars that can run off of

this alternative fuel. Some of these alternatives are different types of biofuel. A

biofuel is a natural alternative to the fossil fuels that are used today. It is made

from living or biological materials that have just died (Biofuel Info). Types of

biofuel include corn ethanol, cellulosic ethanol, and cane ethanol. The type of

biofuel that is most commonly used in the United States is corn ethanol. It is the

most abundant source that can be used as an efficient biofuel. Although sugar

cane can be used to make the most productive biofuel, corn is the most

beneficial for the United States because it can be grown here and it avoids

paying for import taxes on goods from different countries. It also allows the

United States to independently produce a very important product for modern life.

The main reason this biofuel is used is because it emits 51% less

greenhouse gases than gasoline (Biofuel Info). This is because of the more

efficient methods of the production of ethanol. These methods are possible

because of the new technologies. Researcher are constantly trying to find a way

to produce ethanol with a higher alcohol concentration which makes the fuel

more efficient.

Biofuels are made from the fermentation of organic material.

Fermentation is considered to be any process in which large organic molecules

are broken down into simpler molecules. The conversion of sugars or starches to

alcohol is the most widely known type of fermentation (Fermentation).

LaBelle—Miley

This fermentation process is only possible because of yeast. Yeast is

classified as an anaerobic single-celled organism from the Fungi family. When

reproducing, individual yeast cells multiply through a process called budding.

Budding is when a new cell begins as a small bulge on the wall of another cell

(parent cell) and eventually forms into its own entity. Because yeast is anaerobic

it can survive without the presence of oxygen. During such conditions yeasts

converts carbohydrates- starches and sugars- to alcohol and carbon dioxide gas.

During fermentation, certain enzymes in yeast act on starches to break down the

long chainlike molecules into smaller units of sugar, and thus creating the alcohol

(Stairs).

Higher alcohol concentration is directly related to the efficacy of a material

to be used as a biofuel. This is because the higher the concentration of alcohol

in a substance the hotter it will burn. A combustion engine works when the

sudden increase in pressure from the combustion of the fuel expands the

cylinder and pushes the piston, causing the crankshaft to turn. The hotter a

material burns the more combustion is caused in the engine and the more

efficient it will run. In the research being conducted the amount of yeast and the

temperatures that the fermented corn is kept at are altered to maximize the

alcohol concentration and therefore improve it’s effectiveness as a fuel source

(ChemTeacher).

There are two different ways that are used to produce corn ethanol: dry

milling and wet milling.

LaBelle—Miley

Figure 1 is a summary of the dry milling process. Dry milling is the most common

process to make corn ethanol that is used in the United States. This process

requires less energy and it also produces less byproducts. These byproducts are

distiller’s grains, wet stillage, and carbon dioxide. Distiller’s grains and wet

stillage are both given to cattle farmers for feed and the carbon dioxide is sold to

soda companies to use for carbonation. During the first step in the dry milling

process, the corn kernels are ground up into a flour called meal. Then water and

enzymes are added to the meal. This combination is called mash. The enzymes

in the mash break down the starches from the meal and turn them into simple

sugars. The mash is then heated to reduce the amount of bacteria. The mash is

left to cool and once that has happened, yeast is added to start the fermentation

process. The yeast in the mash converts the sugars to alcohol and carbon

dioxide. The mixture is kept at an ideal temperature for the yeast and 40 to 50

hours later, the fermentation process is complete. It is then moved to distillation

columns to separate the ethanol from the mash. Distillation is a method used to

separate substances. The left over mash is the stillage and it is sent to the cattle

farmers. The ethanol from the distillation has about 10-15% gasoline added to it

and is then stored and ready to be shipped off to gas stations (Dry Mill).

Figure 1. Dry milling process

LaBelle—Miley

The wet milling process is a little more intensive process than what is

normally used. The reason that this process may be used is because it produces

more byproducts that can be used. First, the corn kernels are soaked for up to

two days. This starts to break down the bonds between that hold the starch and

the proteins together. After this, the kernels are coarsely ground up to remove

the germ (heart of the kernel) from the rest of the kernel. This germ can be used

to produce corn oil. Once the germ is removed, the corn is more finely ground

up. The fiber is extracted from this using a mill and it is used as a major element

in animal feeds. A centrifuge is used to separate the starch and gluten; the more

dense starch sinks to the bottom. The gluten is taken from the top and also used

for animal feeds. Water and enzymes are added to the starch to ferment it and

turn it into ethanol. Not all the starch can be used so the remaining starch is

used to make high fructose corn syrup. The alcohol is almost ready to be used

after the fermentation process and the carbon dioxide that is produced is sold to

soda companies. The alcohol is then distilled for purification and stored to be

shipped to gas stations (Wet Mill).

The experiment being conducted is a little different from the dry milling

process. First, the corn kernels are mixed with water and blended together until

there are no clumps to make mash. Different amounts of yeast are added to

each mash. Each mash is placed in its own two liter bottle with a balloon

attached on the top to keep the carbon dioxide from leaking out. The balloon fills

with carbon dioxide which helps to visualize that the mash is fermenting. The

two liter bottle is placed into an incubator set at a certain temperature

LaBelle—Miley

corresponding to which trial is being conducted. The amount of yeast that is

added to the mash and the temperature at which the incubator is set are the

variables that are changed with each trial to determine which combination would

yield the highest alcohol concentration. The bottle is left in the incubator for a

week then the liquid is separated from the mash. Then, a hydrometer is used to

test the alcohol concentration of the liquid and it is recorded in a table. A two

factor design of experiment is used to analyze the data.

The impending expiration of gasoline sources has caused much research

in the area of biofuels. This is because biofuels are renewable being made from

living or biological materials that have just died. Corn is the easiest and most

abundant fuel source for the United States to obtain and convert into biofuel

through fermentation and distillation. There are two methods that are used to

make biofuel; wet milling and dry milling. Dry milling is more popularly used

however wet milling produces more beneficial byproducts. The experiment

performed uses different amounts of yeast at different temperatures to see the

effect on the percent alcohol content. The results of this experiment can be

used to make a more efficient and enhanced biofuel.

LaBelle—Miley

Problem Statement

Problem Statement:

What amount of Saccharomyces cerevisiae (yeast) and what temperature

during fermentation would yield the highest alcohol concentration in Zea mays

(corn)?

Hypothesis:

Out of all amounts of Saccharomyces cerevisiae (1, 2, and 3 grams) and

temperatures (22, 25, and 28 °C), 3 grams of yeast at a temperature of 22 °C will

yield the highest concentration of alcohol in fermented corn.

Data Measured:

The dependent variable in the experiment is the concentration in alcohol in

the fermented corn (%), and the independent variables are the amount of yeast

(grams) and temperature during fermentation (°C). The statistical analysis that

will be performed on the data collected was a 2 Factor DOE.

LaBelle—Miley

Experimental Design

Materials:

Active dry yeast (Saccharomyces cerevisiae)Tap water21 half-bushels of corn21 emptied two liter bottlesBalloonsHydrometerBlender250 mL beakerIncubator set at 22 °CIncubator set at 25 °C

Incubator set at 28 °CScaleFunnelStir stick (12 inches)MarkerBowlCupKnifeMesh colander250 mL graduated cylinder

Procedures:

1. In a cup, measure the amount of yeast needed on a scale according to

what trial is being conducted

2. Fill the cup about half way up with water

3. Let the yeast stay in the water for about 15 minutes

4. Peel off the husks of half of a bushel of corn (four ears)

5. Cut the kernels off the corn with a knife and place them in a bowl

6. On a scale, measure one part water to one part corn

7. Put the water and the corn into the blender

8. Blend the mixture until fully blended

9. Strain mixture with a colander into a 250 mL beaker

10.Pour the liquid that was strained into the 250 mL graduated cylinder so

that it is almost full

11. Use the hydrometer to measure the alcohol content

LaBelle—Miley

12.Record the value in the table

13.Blend the liquid and the corn mixture back together

14. Add the yeast to the mixture and stir with a stir stick

15.Pour mixture into a two liter bottle using a funnel

16.Label the bottle with the amount of yeast used and the alcohol content

17.Place a balloon on the bottle

18. Place the bottle into the correct incubator

19. After four days, remove the bottle from the incubator

20. Strain the liquid out of the mixture with a colander into a 250 mL beaker

21.Pour the liquid into a graduated cylinder so that it is almost full

22.Use the hydrometer to measure the percent of alcohol in the liquid

23. Record the value in the table

24. Repeat steps 1-23 for each combination of temperature and amount of

yeast

Diagram:

Figure 1. Materials

LaBelle—Miley

Data and Observations

Data:

Table 1Percent Alcohol Content of the Fermented Corn

Trial (+/+) (+/-) (-/-) (-/+)1 7 8 5 52 7 7 5 53 8 8 6 6

Table 2 Percent Alcohol Content of the Standard Trials

Trial Standard1 62 63 74 65 56 67 68 69 6

Table 3Values of Temperature and Yeast

Temperature (°C) Amount of Yeast (g)- Standard + - Standard +

22 25 28 1 2 3

LaBelle—Miley

Observations:

Table 4Observations

Date ObservationOct 22 9 standard trials are set up. Corn and yeast blends are consistent.Oct 26 Corn and yeast blend produced CO2 which can be seen by the

balloon inflating (see Figure 1).Oct 29 All (+,-) trials produced CO2 and the balloons inflated.Nov 2 Only 3 (+,+) trials produced CO2. The balloon on the Trial 2 bottle

did not inflate resulting in a redo of this trial.Nov 6 All (-,-) trials produced CO2 and the balloons inflated.

Nov 10 All (-,+) trials produced CO2 and the balloons inflated.Nov 13 Trial 17 (-,+) blend seemed more separated than the rest, but still

produced CO2 and stayed consistent with rest of trials.

Figure 1. Two of the Standard Trials

LaBelle—Miley

Data Analysis and Interpretation

Data Analysis:

Experiment: The Effect of Yeast and Temperature on the Alcohol Content of Corn after Fermentation

Response Variable: Alcohol Content (%)

Predictor Variable: Temperature

Predictor Variable: Amount of Yeast

Table 1. High and Low Values

Temperature (°C) Amount of Yeast (g)- Standard + - Standard +

22 25 28 1 2 3

Table 2.Design of Experiment ResultsOrder Runs Result Order Runs Result Order Runs Result

1 Stand 6 1 Stand 6 1 Stand 65 + + 7 5 + + 7 6 + + 86 - - 5 2 - - 5 3 - - 64 Stand 6 4 Stand 5 4 Stand 62 + - 8 3 + - 7 2 + - 83 - + 5 6 - + 5 5 - + 67 Stand 7 7 Stand 6 7 Stand 6

Table 3.Standards

Nine Standards6 6 7 6 5 6 6 6 6

LaBelle—Miley

0 1 2 3 4 5 6 7 8 90

2

4

6

8

10

12

Trial

Alco

hol C

onte

nt (%

)

Figure 1. Standard Runs

Table 4.Averages of Data

RunsFirst DOE Second

DOE Third DOE AverageTemperature(°C)

Amount of Yeast (g)

+ + 7 7 8 7.33- - 5 5 6 5.33+ - 8 7 8 7.67- + 5 5 6 5.33

Grand Average: 6.145

In Table 1, the high and low values that were used for temperature

measured in degrees Celsius (°C), and yeast measured in grams (g). Table 2

shows the results for the three trials of the experiment, with the combinations of

high and low values for the temperature and amounts of yeast. Table 3 shows

the results of the nine standards, which are graphed in Figure 1. Table 4 shows

the all of the averages of the three designs of experiment (DOE).

LaBelle—Miley

-1 10

2

4

6

8

10

5.33

7.5

Temperature (°C)

Alco

hol C

onte

nt (%

)

Figure 2. Effect of Temperature

Table 5.Effect of Temperature

Temperature (°C)(-) 22 (+) 28

5.33 7.335.33 7.67

Avg = 5.33 Avg = 7.5

The effect is 2.17 units.

Figure 2 and Table 5 show the effect of temperature on the alcohol

content in fermented corn. When the temperature was at the high, 28°C, the

average alcohol content was 7.5. When the temperature was at the low, 22°C,

the average alcohol content was 5.33. The effect of temperature was 2.17 units.

On average as temperature increased, the alcohol content increased by 2.17

units.

Amount of Yeast (g)(-) 1 (+) 3

5.33 7.337.67 5.33

Avg = 6.5 Avg = 6.33

LaBelle—Miley

-1 10

2

4

6

8

10

6.5 6.33

Amount of Yeast (g)

Alco

hol C

onte

nt (%

)

Figure 3. Effect of Yeast The effect is -0.17.

Figure 3 and Table 6 show the effect of the amount of yeast on the alcohol

content of fermented corn. When the amount of yeast was at the high, 3 grams,

the average alcohol content was 6.33. When the amount of yeast was at the low,

1 gram, the average alcohol content was 6.5. The effect of the amount of yeast

was -0.17 units. On average as the amount of yeast increased, the alcohol

content decreased by 0.17 units.

Interaction Effect (Temperature and Yeast)

Amount of Yeast (g)Low (1) High (3)

Tem

pera

ture

(°C

)

Solid Segment

High (28) 7.67 7.33

Dotted Segment

Low (22) 5.33 5.33

LaBelle—Miley

-1 10

2

4

6

8

10

7.67 7.33

5.33 5.33

Amount of Yeast (g)

Alco

hol C

onte

nt (%

)

Figure 4. Interaction of Temperature and Yeast

Table 7.Interaction of Temperature and Yeast

Slope of the segment of Temperature (+) minus Slope of the segment of

Temperature (-) gives the Effect (Temperature vs Yeast) = -0.17 - 0 = -0.17

Table 7 shows the interaction of temperature and yeast. The interaction

effect is shown in Figure 4. There may be interaction between temperature and

yeast because they do not meet on the graph, but their slopes are not parallel.

Taking the slope of the segment of temperature high minus the slope of the

segment of temperature low gives the effect of -0.17 units.

To calculate the prediction equation, each value effect is divided by the

range of standards. The range of standards is 2 (7-5=2). In order for an effect to

be significant, the absolute value of the quotient must be greater than or equal to

2. Only the effects that are significant are used in the prediction equation.

According to this, none of the effects are deemed significant.

Prediction Equation:

Y=6 . 415+2 . 172

∗t+−0 . 172

∗y+−0 . 172

∗yt+ ital noise

LaBelle—Miley

Y=6 . 415+ ital noise

LaBelle—Miley

Conclusion

The hypothesis was rejected. Out of all amounts of Saccharomyces

cerevisiae (1, 2, and 3 grams) and temperatures (22, 25, and 28 °C), 3 grams of

yeast at a temperature of 22 °C did not yield the highest concentration of alcohol

in fermented corn. After a 2 Factor DOE statistical analysis it was concluded that

neither temperature during fermentation or amount of yeast had a significant

effect on the alcohol content in corn after fermentation.

These results occurred because the only factor that will have a significant

effect on the amount of alcohol in a material is the amount of sugar in a material

(Alba-Lois). Temperature will only speed up the process. Chemical reactions

within yeast are facilitated by enzymes, which are large organic catalysts. Each

enzyme has an optimal temperature range. At too of low temperatures, 0-10 °C,

yeast will not grow. At temperatures 10-37 °C yeast will grow and multiply, faster

at higher temperatures with an optimal growth at 30 °C (for the Saccharomyces

cerevisiae species). At higher temperatures the cells become stressed, meaning

that their content becomes damaged and can not be repaired. At these high

temperatures, above 50 °C, the cells die (Curry). Because the range of

temperatures in this experiment was relatively small the temperature did not have

an effect. However if the range would have been 0°C to 50°C temperature would

have most likely had an effect on the alcohol content of the corn after

fermentation.

LaBelle—Miley

The amount of yeast had no effect because yeast can only convert the

sugars that are present in the mixture and after these sugars are converted can

do no more. As long as there is enough yeast to convert all of the sugars in a

mixture adding more yeast will have no affect because there will be no sugars left

to convert into alcohol, only left over yeast (Janson).

The results of this experiment agree with the current work in the field.

Temperature is only known to speed up the effects of fermentation, not actually

causing the alcohol concentration to be higher. The type of yeast used will cause

the optimal temperature to vary because each species of yeast has slightly

different ideal conditions. It was also known that yeast has little or no effect after

an initial needed amount of yeast and that extra yeast will not increase alcohol

content. The results of this experiment will impact that scientific community

because it gives evidence for commonly known theories.

There were a few design flaws during experimentation. First off, the

incubator was not at the exact temperature that it was supposed to be at. Due to

the dials on the incubator, it was hard to know what to set each dial at to get the

correct temperature. The temperature was sometimes a few tenths of a degree

off, but was not deemed significant because temperature only speeds up the

process of fermentation. Sometimes when the yeast was set in the water to

rehydrate, it was left for a few minutes longer than it should have been. This was

also insignificant because the yeast was not affected by it. During the

fermentation, some of the balloons on top of the bottles expanded and some of

them did not. The balloons that did not expand may have let out some carbon

LaBelle—Miley

dioxide. If a balloon did not inflate, that trial was thrown out and redone. Also,

some of the corn may have been blended more than other corn. This did not

seem to have an effect on the alcohol content of the corn either.

Further experimentation could be done with different types of yeast. The

different types of yeast would be added to the corn and they would all be set at

the same temperature and fermented for the same time. The alcohol content

would then be found to see which type of yeast could produce the highest. Also,

different types of corn could be used. Potentially, the corn with the highest sugar

content would produce the highest alcohol content, so that could be tested.

Instead of using corn, different plants could be used. These different plants have

different sugar levels and this could cause higher alcohol content. Experimenting

with different plants could help find an abundant source that would be more

efficient.