Which bacteria or combination of bacteria would be most successful in the

breakdown of bamboo into simple sugars?

Tristan MacAneney

South Side High School

Science Research I

Museum of Natural History

Young Naturalist Competition National Finalist 2012

Introduction:

With a quickly developing, dangerous climate change, and an increase in the cost

of oil, researchers are competing with time to develop environmentally friendly biofuels.

Ethanol, an alcohol that can be produced from a wide variety of plant materials, is used as

an additive to gasoline, a liquid fuel in motor vehicles (Cellulosic Ethanol Feedstock).

Due to an increasing demand for cheaper and cleaner fuel, researchers are hoping to

supplement gasoline with ethanol. Cornstarch and sugarcane are the two main feedstocks

used, producing starch- and sugar-based ethanol. Cellulose, another type of plant

material, can also be produce ethanol, but doing so requires additional processing to

further break down cellulosic materials into sugars. Ethanol produced from cellulose is

referred to as cellulosic ethanol. Until now, efforts to calculate which crops are more

environmentally friendly have focused solely on the amount of greenhouse gases emitted

when the biofuel is burned (Jha, 2008). In contemporary research, scientists have found

that using biofuels made from corn, sugar cane, soy, and other edible plants is largely

impractical and could have environmental implications that are just as detrimental as

burning fossil fuels. Burning biofuels emits fewer greenhouse gases than burning fossil

fuels, however, the production of biofuels requires biodiversity loss and destruction of

farmland (Jha, 2008).

With an increase in the number of motor vehicles being used today, the U.S.

consumes about 130 billion gallons (almost 500 billion liters) of gasoline per year.

Gasoline is known as an aliphatic hydrocarbon, meaning it is made up of molecules

composed of only hydrogen and carbon, arranged in chains. Gasoline molecules have

seven to eleven carbons in each chain. A common configuration of a gasoline chain looks

like this: H-C-C-C-C-C-C-C-H. When gasoline is burned with sufficient oxygen under

ideal conditions, carbon dioxide, water, and heat are produced as byproducts. (How

Gasoline Works). However, the internal combustion engine in cars is not perfect and

produces carbon monoxide (a poisonous gas), nitrogen oxide (the main source of urban

smog) and unburned hydrocarbons (the main source of urban ozone depletion). Gasoline

is made from mostly carbon atoms, so a gallon of gas may release up to 6 pounds of

carbon into the atmosphere. Because the 6 pounds of carbon come out as an invisible gas

(carbon dioxide), it is not very recognizable. The ultimate effects of these greenhouses

gasses including carbon are unknown, but early research has linked these greenhouses

gasses to global warming and ozone depletion. Unlike gasoline, ethanol is an oxygenated

fuel that contains 35% oxygen, which reduces particulate and NOx emissions from

combustion (Badger 2002).

Bamboo has been recently researched as a renewable bioenergy crop, because of

certain valuable traits that are absent in more commonly used crops such as corn,

sugarcane, and soy. Since it is a quickly growing plant and can grow in dense conditions,

bamboo is considered one of the best renewable resources on the planet (wizeGEEK,

2012). It is now being considered for use in several bioenergy conversion processes,

including cellulostic ethanol conversion. Rather than producing ethanol synthetically

from petroleum, the production of ethanol by the microbial conversion of biomass

materials through fermentation is less harmful to the environment (Badger 2002).

Fermentation involves microorganisms, including bacteria, which use the fermentable

sugars from biomass for food, and in the process produce ethanol as a byproduct (ABC's

of Biofuels, 2011). These microorganisms use a 6-carbon sugar for food, with glucose

being the most common. With that said, biomass materials containing high levels of

glucose or precursors to glucose are the easiest to convert to ethanol. Because glucose is

part of the human food chain, it would be more practical to take advantage of bamboo

and other biomasses that are not traditionally consumed by humans, for the production of

biofuels. Being a cellulostic material, bamboo happens to contain sugars within it’s

cellulose and hemicellulose, but is more difficult to biochemically convert into ethanol

than starch- and sugar-based feedstocks (Cellulosic Ethanol Feedstocks, 2009).

Cellulostic plants are comprised of lignin, an organic substance binding the cells

(About Lignin), which encloses the cellulose and hemicellulose molecules, making the

sugar difficult to reach (Badger 2002). Cellulostic plants such as bamboo, however, are

more abundant than other crops, and can be used to produce more substantial amounts of

ethanol to meet U.S. fuel demand. They can also be grown on marginal lands that are not

suitable for other crops. Less fossil fuel energy is required to grow, collect and convert

these types of plants to ethanol. The Fischer-Tropsch Process is a multistep process,

which is essentially a catalyzed chemical reaction that takes a gas mixture of carbon

monoxide and hydrogen, and converts it into liquid hydrocarbons. The idea that this

process may be helpful in the breakdown of bamboo has come into play, but faces many

impracticalities, one being that it must take place in a strict temperature between 150-200

degrees Celsius (Bamboo Biofuel and Biodiesel 2012). The focus of research by the

U.S. Department of Energy has been to economically develop processes to break down

components of cellulostic biomass, including lignin, which would result in easier ethanol

conversion. At the National Meeting & Exposition of the American Chemical Society in

Denver, researchers reported that microbes found in the feces of pants, could possibly

optimize the breakdown of raw plant materials used to make biofuels (Pappas, 2011).

Research done at Mississippi State University, by researcher and biochemist, Ashli

Brown, suggests that under a stable environment, the bacteria found in panda feces can

covert 95 percent of plant biomass into simple sugars, which would then be converted

into ethanol (Pappas, 2011). Thus, the current need for high heat, harsh acids or high

pressures used to produce biofuels, would be eliminated. The question is, which

bacteria or combination of bacteria would be most successful in the breakdown of

bamboo into simple sugars?

The purpose of this project is to determine whether or not bamboo can serve as a

sufficient source of ethanol production. In order to carry out the steps needed to produce

ethanol, I first needed to figure out which bacteria would be most successful in the

breakdown of bamboo cellulose into more simple sugars. To test this question, Bacillus

subtillis (B. subtillis), Escherichia coli (E. coli), and a combination of both B. subtillis

and E. coli were added to bamboo samples and incubated over a one-week period. I

predicted that the combination of both E. coli and B. subtillis would be most successful in

the breakdown of bamboo into simple sugars because they are two very common bacteria

found within the digestive system of many animals including humans. Bacteria thrive

best as they break down material. After the one-week period, I tested percent

transmittance of the bacteria after they were added to bamboo and compared those

readings to the original percent transmittance readings of the bacteria before the

experiment. We can infer that if the percent transmittance decreases, then the bacteria are

thriving, and therefore breaking down the bamboo plant matter.

When converting more commonly used crops such as corn, enzymes are used to

convert starches to simple sugars and yeasts are used to ferment the sugars into ethanol.

Cellulosic biomass contains sugars as well, but because of the lignin in the stems, they

are much harder to release than those in starchy biomass. To complicate matters, the

process of releasing the sugars produces by-products that inhibit fermentation, and some

sugars from cellulosic biomass are difficult to ferment.

This fermentation method generally uses three steps:

(1) The formation of a solution of fermentable sugars

6 CO2 + 6 H2O + light → C6H12O6 + 6 O2

(2) The fermentation of these sugars to ethanol

C6H12O6 → 2 C2H5OH+ 2 CO2 + heat

(3) The separation and purification of the ethanol, usually by distillation or

combustion

C2H5OH + 3 O2 → 2 CO2 + 3 H2O + heat

(Badger, 2002)

Materials and Methods:

Materials:

• 1 bamboo plant

• 40 test tubes

• Aluminum foil

• Tuttnauer 2540 M autoclave

• Pipettes

• Scout Pro 400 gram OHAUS electronic scale

• Rubber gloves

• B. subtillis bacteria

• E. coli bacteria

• Spectronic 20 Bausch and Lomb spectrophotometer

• Cheese Cloth

Methods:

In order to complete this experiment, bamboo must first be cut up and divided

into two different beakers. The first beaker contains the stems of the bamboo and the

second beaker contains the leaves of the bamboo.40 test tubes will be wrapped in

aluminum foil and autoclaved at 121 degrees Celsius and at 10 ms-1. Mix the tube

beakers of stems and leaves, and using the scout pro 400g- OHAUS electronic balance

scale, measure out 7.12 grams of bamboo. This bamboo should then be divided equally

among the 40 crucibles, at .18 grams per test tube. Unwrap the 40 test tubes and divide

them into 4 different groups, with 10 test tubes in each group. The first group will be for

the B. subtillis and should be labeled with a “B,” and numbered 1 through 10. The

second group will be for the E. coli and should be labeled with an “E” and numbered 1

through 10. The third group will be for the combination of both B. subtillis and E. coli

and should be labeled with a “C” and numbered 1 through 10. The last group of test

tubes should be split up into 3 groups of 3, leaving 1 extra test tube that will not be used

in the experiment. These 9 test tubes will be used for the control of the experiment. The

first 3 test tubes will be for the B. subtillis control and should be labeled “BC” and

numbered 1 through 3. The next 3 test tubes will be for the E. coli and should be labeled

“EC” and number 4 through 6. The last three test tubes will be for the combination

control and should be labeled “CC” and numbered 7 through 10. Next, add .18 grams of

bamboo to each of the first 30 test tubes, leaving the last 9 test tubes empty. The

spectrophotometer will then be used to calculate the percent transmittance for each

bacterial broth. Mix even parts of B. subtillis and E. coli in a separate beaker. Pipettes

will then be used to add bacterial broth to the test tubes and fully submerge the bamboo.

Add B. subtillis to the test tubes labeled “B” and “BC.” Add E. coli to the test tubes

labeled “E” and “EC.” Add the combination of B. subtillis and E. coli to the test tubes

labeled “C” and “CC.” Cover the top of each test tube with aluminum foil and allow the

bacteria to incubate over a one-week period. After 7 days, remove the test tubes from the

incubator and remove the aluminum foil from the top. Next, use cheese cloth to strain

out the bacteria from test tube “BC1” into a cuvette. Leave the bamboo in the test tube

for later use, and using the mass spectrophotometer, take the reading of percent

transmittance from the bacteria in the cuvette. Next, dispose of the bacteria and wash out

the cuvette. Repeat for test tubes BC2 through CC9.

Data:

The data for this experiment was collected and organized into the following tables

and charts. The data was also analyzed using ANOVA single factor and T-tests. The

table below (see Table 1) shows the percent transmittance of each bacterium’s trial as

well as the combination and control. It was found that the combination of bacteria had

the most prolific growth with a mean percent transmittance of 3.9%, while the E. coli and

B. subtillis values were 7.1% and 5.5%, respectively. All bacteria had much lower

transmittance values than their controls (see Figure 2).

Trial B. subtillis E. coli Combination B. subtillis Control E. coli Control Combination Control 1 3 20 5 78 84 78 2 12 13 15 92 82 82 3 3 10 0 85 80 90 4 6 9 13 5 14 7 0 6 2 4 0 7 6 0 0 8 2 2 1 9 5 1 0

10 2 5 5 Mean 5.5 7.1 3.9 85 82 83.33333333 SD 4.2752518 6.154 5.70477383 7 2 6.110100927 95% CI 2.6497798 3.815 3.53579048 7.921100139 2.263171468 6.914103042

The ANOVA test shows that the p-value was 4.11 x 10-26%. The T-tests for B.

subtillis & it’s control (no bamboo), E. coli and it’s control, and the bacterial combination

and it’s control were 5.44 x 10-11%, 4.80 x 10-10% and 3.37 x 10-10%, respectively.

Conclusion:

Which type of bacteria would be most successful in the breakdown of

bamboo? I predicted that the combination of both E. coli and B. subtillis would be most

successful because of their common presence in the digestive system of most animals.

Figure 2: Data table showing mean, standard deviation and 95% confidence intervals for bacterial growth with and without the presence of bamboo.

Specific cases from the combination trials compared to the B. subtillis trials and E. coli

trials proved my prediction correct. For example, on trial 5, there was a reading of 0%

transmittance for the combination, but 7% transmittance for E. coli and 14%

transmittance for B. subtillis. Because of the low percent transmittance reading for the

combination, we can infer that the bacteria in that test tube are thriving best, and

therefore working to break down the bamboo. In trial one, the B. subtillis control

(without bamboo) had 78% transmittance, but the regular B. subtillis trial group had a

reading of 3% transmittance, which further demonstrates bacteria thriving under the

presence of bamboo and working to break down the bamboo into simple sugars. The

results show that a combination of bacteria would be most successful in the breakdown of

bamboo into simple sugars. Although I was able to see which bacterium thrived best, I

was unable to get the readings for the amount of cellulose that was actually broken down

into simple sugars. Although it is inferred, this was still a limiting factor that affected the

experiment. Recent studies by the U.S. Department of Energy (DOE) suggest however,

that by 2030, the DOE will have the technology necessary to convert cellulosic material

into ethanol. Resources such as bamboo will then be used to produce sufficient ethanol –

about 60 billion gallons/year – to displace about 30% of our current gasoline

consumption by 2030 (Research Advances NREL Leads the Way).

References

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