Research Paper-Google Science Fair

7/30/2019 Research Paper-Google Science Fair

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Investigating the Use ofAnaerobic Fermentation

on Pretreated Biomass toStreamline Bio-fuel

Production


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Table of ContentsPurpose ____________________________________________________________________________ 1

Introduction ________________________________________________________________________ 1

Figure 1: World Ethanol Production: 2007-2011 __________________________________________ 1

Figure 2: Greenhouse Gas Reductions __________________________________________________ 3

Figure 3: Biofuels Lifecycle Emissions ___________________________________________________ 3

History _____________________________________________________________________________ 4

Figure 4: Ethanol Net Returns and Corn Prices ____________________________________________ 5

Methods and Materials ________________________________________________________________ 6

Acid pretreatment__________________________________________________________________ 6

Acid hydrolysis Determination of Klason lignin __________________________________________ 6

Figure 5: Mixing Sulfuric Acid into Biomass ______________________________________________ 7

Figure 6: Verifying Neutral pH of Klason lignin ____________________________________________ 8

Figure 7: Formula for Determining Klason Lignin __________________________________________ 8

Determination of Carbohydrate Content ________________________________________________ 9

Figure 8: Glucose and Xylose Percentages ______________________________________________ 10

Introduction of Bacteria to Facilitate Ethanol Production __________________________________ 10

Figure 9: Boiling to Remove Oxygen ___________________________________________________ 11

Fi 10 Addi S l i A l d M di 12
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Figure A3: Trace Element Formula _____________________________________________________ 2

Figure A4: Selenium Tungsten Solution ________________________________________________ 2

Figure A5: Vitamin Formula __________________________________________________________ 2

Figure A6: Sodium Sulfide Solution _____________________________________________________ 2

Figure A7: Glucose and Xylose Percentages ______________________________________________ 2

Figure A8: HPLC Results Ethanol Content ________________________________________________ 2

Figure A9: Klason Lignin Content ______________________________________________________ 2

Figure A10: Average Ethanol Content ___________________________________________________ 2

Figure A11: Comparison of Ethanol Content 2012 and 2013 _________________________________ 2
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PurposeThe purpose of this investigation was to determine if anaerobic fermentation

could be utilized as a feasible method to produce ethanol biofuel; and which of the

bacteria in the trials, Clostridium thermocellum, Clostridium thermolactium, or a co-

culture using equal parts of both microorganisms would produce the most ethanol.

IntroductionThe United States is the largest producer of ethanol fuel, producing 15.2 billion

gallons in 2012. The USA and Brazil produce most of the worlds ethanol fuel, as

shown in Figure 1: (Source: F. O Licht, cited in Renewable Fuels Association, Ethanol

Industry Outlook 2008-2012 reports.)


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The USA uses corn while Brazil uses sugarcane as primary glucose sources to

produce ethanol. Brazil has aggressively promoted the use of ethanol fuel; Brazilian

law has required an ethanol-gasoline blend since 1986 and a 25-percent blend of

ethanol to gasoline since 2007, although this dropped to 20% in 2010 because of

diminished production of sugar cane crops. In the United States, Portland, Oregon (in

July of 2006) became the first American city to require that all gasoline sold within the

city limits be blended with at least ten percent ethanol. Although federal clean air laws

do not require the sale of E10 ethanol blends, they do mandate specific amounts of

ethanol be used in each state, to be decided by the state. This requirement may be met

with E10, E15, E20, or E85. In 2011, the 133.93 billion gallons of gasoline (3.19 billion

barrels) consumed in the United States contained about 12.87 billion gallons of ethanol,

accounting for nine percent of the volume of gasoline consumed. (U.S. Energy

Information Administration)

Ethanol reduces gasoline prices: After subtracting the cost of subsidies, ethanol


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In 2011, the production and use of 13.9 billion gallons of ethanol in the U.S.

reduced CO2-equivalent greenhouse gas emissions by 25.3 million tons, the equivalent

to removing four million vehicles from America's roadways. (Source: Argonne National

Laboratory's GREET Model).

Figure 2: Greenhouse Gas Reductions


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Ethanol feeds livestock: About one third of the cellulose can be recovered to

produce animal feed.

HistoryHenry Fords first vehicles ran on ethanol, as did most of the farm equipment of

the time. Because it was cheaper than gasoline, it remained the fuel of choice until

1901 when the Texas oil fields were discovered, which dropped the cost of gasoline

below ethanol.

In the mid-seventies, the octane-boosting lead additive used in gasoline was

discovered to be a dangerous pollutant, so it was replaced with methyl tertiary-butyl

ether (MTBE). Due to studies that discovered MTBE in groundwater and showed it to

be carcinogenic, ethanol was substituted as an oxygenator in gasoline starting with

California in 2003. Since then, E10 gas (10% ethanol-gasoline blend of fuel) has

become the conventional fuel and gasoline without ethanol is now atypical and is

difficult to find.


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Cellulosic ethanol could be considered a stable source because it is created

solely from waste. Today, most of the ethanol biofuel created in the United States is

made from corn kernels. Aside from the fluctuating costs, this creates a conflict with

food production, and raises questions about the advisability of using arable land for

anything but food when there is a world need for more food production. This is where

cellulosic ethanol can step in to fill the void with a desperately needed alterative to

ethanol produced from corn. If the obstacles to cheap, clean cellulosic ethanol

production can be resolved, then a superior, renewable energy source will be available

Figure 4: Ethanol Net Returns and Corn Prices


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Cellulosic ethanol production has remained a much-studied science because the

rewards can be substantial. According to former Energy Secretary, Samuel Bodman,

"Cellulosic ethanol contains more net energy and emits significantly fewer greenhouse

gases than ethanol made from corn"

Methods and MaterialsThis research examined the residue from a Zea mays L. (corn) crop. Called,

corn stover, it consists of the plants left in the field after harvesting, which includes the

leaves, stalks, husks and cobs.

Acid pretreatment

The biomass samples were cut into small pieces and dehydrated in a drying oven set at

149 Celsius. Once dried, the biomass was crushed in a grinder until the particles were

approximately the size of 50 microns. A five-gram sample of the biomass was collected

and a one-percent sulfuric acid (H2SO4) solution was introduced into it. The treated

biomass was placed in a metal cylinder that had been forcefully tightened to allow

pressure to build up in it It was placed in a 120 degree Celsius oil bath for 45 minutes


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After the acid was thoroughly mixed with the biomass; the beaker, with the glass

rod, was placed in a vacuum desiccator for 15 minutes to facilitate wetting and

dispersion. After dispersion, the beaker was covered with aluminum foil and placed in a

water bath at 30 degrees Celsius for 60 minutes and the biomass was stirred frequently

with the glass rod. The beaker was removed and 84 mL of deionized water was added

to dilute the concentration of H2SO4 to 3.0 percent. Four calibration solutions were

prepared containing five monosaccharides (glucose xylose galactose arabinose and

Figure 5: Mixing Sulfuric Acid into Biomass


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acid-insoluble lignin (Klason lignin). The lignin was washed with hot water to remove

the acid, and a neutral pH was verified with a pH paper.

The filter papers with the lignin were dried in an over at 105 Celsius overnight or

to a constant weight, then cooled in a desiccator and weighed. After the samples

l d t t t th filt t t i i th h id d th id

Figure 6: Verifying Neutral pH of Klason lignin


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Determination of Carbohydrate Content

The filtrate, containing the monosaccharides and the acid-soluble lignin, was

tested to determine the percentages of glucose and xylose using the High-Performance

Liquid Chromatography (HPLC): To begin, the HPLC was calibrated by running pure

samples of glucose and xylose.

To prepare the filtrate samples to be run through the HPLC, the liquid from each

sample was filtered through a .45 m membrane into a small glass vial that is specially

designed for the HPLC. The samples were placed in the HPLC at specific locations that

identified them by untreated 1 or 2 or treated 1 or2, samples; and processed to

determine the glucose and xylose content.


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TreatedSample One

TreatedSample Two

UntreatedSample One

UntreatedSample Two

Glucose

Percentage 48.4 49.2 33.1 33.8

XylosePercentage

17.3 19.1 16.3 13.5

AveragePercentage

Glucose48.8 33.5

Average

PercentageXylose

18.2 14.9

Introduction of Bacteria to Facilitate Ethanol Production

The media for cultivating the bacteria was created using the lists of chemicals in

Appendices A1 through A6. Utilizing a scale precise to 1/10000th of a gram, nine, .002

gram specimens of pretreated biomass were isolated and placed in nine, ten mL, sterile

serum bottles. Six more bottles had the same amount of untreated biomass inserted

into them. Next, five milliliters of the media was introduced into each bottle, and they

were separated into three batches of five each, three with treated and two with

Figure 8: Glucose and Xylose Percentages


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The bottles were removed from the heat source and cooled to 22 degrees

Celsius using a nitrogen/carbon dioxide gas. Using the crimping tool, the crimp seals

were seated onto each bottle. This had to be done promptly to avoid oxygen reentering

the media. The samples were placed in the autoclave for 20 minutes at 20 pounds per

square inch and 121 degrees Celsius to remove any unwanted organisms, and the

samples were cooled to 22 degrees Celsius. To create the ideal environment for

culturing the bacteria, a hypodermic syringe was utilized to introduce the trace element

Figure 9: Boiling to Remove Oxygen


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Using a hypodermic syringe, the following bacteria was added to the fifteen

samples: .5 ml of Clostridium thermocellum was added to five samples, .5 ml of

Clostridium thermolactium was added to another five samples, and .25 ml of Clostridium

thermolactium and .25 ml of thermocellum (co-culture) was added to final five samples.

These strains of bacteria were used because they can live at higher temperatures than

most bacteria.

Figure 10: Adding Solutions to Autoclaved Media


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locations that identified them by material and as a, b, or c samples; and processed

to determine the ethanol content.

Using Bacterial Cellulose Hydrolysis to Create Ethanol


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ResultsAfter performing bacterial cellulose hydrolysis and fermentation on the

Clostridium thermocellum strain, the Clostridium thermolactium strain, and the co-

culture, which consisted of equal parts of the two strains; and, after examining the High

Performance Liquid Chromatography (HPLC) test results, it was concluded that the

most viable bacteria choice for large-scale ethanol production was the co-culture. This

was based on high ethanol content. See Figures 12, A7, and A8.

The five samples of the Clostridium thermolactium strain performed poorly

compared to the Clostridium thermocellum, producing significantly less ethanol than

either the Clostridium thermocellum or the co-culture.

Clostridium Thermocellum efficiently degrades hexoses, monosaccharides with

six carbon atoms, while Clostridium thermolactium proficiently degrades pentoses,

monosaccharides with five carbon atoms. Therefore, the co-culture performed better

than either of the single strain cultures, since it was able to convert most of the simple

sugars.


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Comparison of Enzymatic Hydrolysis (2012) and Bacterial Hydrolysis (2013)

Enzymatic Hydrolysis Bacterial Cellulose Hydrolysis

Biomass

(2011-2112)NaOH PretreatmentAverage Ethanol %

(a and b)v/v

(2011-2012)H2SO4 PretreatmentAverage Ethanol %

(a and b)v/v

(2012-2013)Clostridium

ThermocellumAverage Ethanol %

(a,b,c)v/v


ThermolactiumAverage Ethanol %

(a,b,c)v/v

(2012-2013)Co-culture

Average Ethanol %(a,b,c)

v/v

CornStover 5.135 8.994 7.18 5.08 14.55

DiscussionThe focus of this study was investigating the use of bacteria to break down

cellulose, retrieve sugars, and ferment them into ethanol. This streamlined, one-step

approach could be a viable method to convert cellulosic biomass into renewable fuel.

These studies are vital because finding the best process is imperative to a cost-effective

cellulosic ethanol program.

Figure 12: Average Ethanol Content 2012-2013


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the sugars is still protein-rich and can be used as livestock feed, which provides another

revenue source to offset the cost of ethanol production.

Another important aspect of lowering costs is finding better enzymes. Studies

are being made on the San Diego Supercomputer Center (SDSC) to examine the

structures of biomass products and enzymes at the molecular level to fully understand

how enzymes release glucose, with a goal of finding and/or creating more efficient and

cheaper enzymes. This is an important key to cost-effective cellulosic ethanol

production, since the cost of enzymes is a significant factor in glucose recovery.

The U.S. Department of Energys National Renewable Energy Laboratory

(NREL) has just forged an agreement with Johnson Matthey, chemical manufacturer, to

commit five years and seven million dollars in a collaboration to find a catalyst that will

lower the cost of producing cellulosic biofuels. Many such efforts are being undertaken

today because the rewards of success would be enormous.

Last year this study discussed corn stover extensively because it comprises 75%

of agricultural waste and is readily available. The drawbacks were primarily soil loss


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released a sustainable retention map, which shows areas of the country that require

high retention and areas that are sustainable with low retention. See Figure 13.


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AcknowledgmentsI thank Dr. Ulrike Tschirner, from the University of Minnesota, for her

generous and steadfast assistance with equipment and materials,

with bacteria, and with informed counsel.


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ReferencesEnergy Efficiency and Renewable Energy, 2011, Crop Residues and AgriculturalWasteshttp://www1.eere.energy.gov/biomass/pdfs/btu_crop_residues.pdf

Osborne, Stefan, 2007, Energy in 2020: Assessing the Economic Effects ofCommercialization of Cellulosic Ethanolhttp://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ia

n_002699.pdf

Milbrandt, Anelia, 2006, Geographic Perspective on the Current Biomass ResourceAvailability in the United States, NRELhttp://www.nrel.gov/docs/fy06osti/39181.pdf

Davis, John, 2009, International Energy Agency (IEA)http://domesticfuel.com/2009/10/13/iea-global-biofuel-production-to-rise-big-by-2012/

Glossary of Biomass Termshttp://www.nrel.gov/biomass/glossary.html

Lane, Jim, 2010, EPA Confirms Tiny Cellulosic Biofuels Mandate for 2011http://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011

Hayes, Dermot, 2012, New University Study: Ethanol Reduced Gas Prices by Morethan $1 in 2011http://www.ethanolrfa.org/
http://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdfhttp://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdfhttp://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdfhttp://www.nrel.gov/docs/fy06osti/39181.pdfhttp://domesticfuel.com/2009/10/13/iea-global-biofuel-production-to-rise-big-by-2012/http://www.nrel.gov/biomass/glossary.htmlhttp://www.nrel.gov/biomass/glossary.htmlhttp://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011http://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011http://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011http://www.ethanolrfa.org/http://www.ethanolrfa.org/http://www.ethanolrfa.org/http://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011http://www.renewableenergyworld.com/rea/news/article/2010/12/epa-confirms-tiny-cellulosic-biofuels-mandate-for-2011http://www.nrel.gov/biomass/glossary.htmlhttp://domesticfuel.com/2009/10/13/iea-global-biofuel-production-to-rise-big-by-2012/http://www.nrel.gov/docs/fy06osti/39181.pdfhttp://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdfhttp://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdf


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Page A1 ofA7

Appendices

Basal Medium

Chemical Formula Required Grams (g)Sodium Chloride NaCl 10.000

Magnesium MgCl2.6H2O 0.500

Potassium Dihydrogen Phosphate KH2PO4 0.200

Ammonium Chloride NH4Cl 0.300

Potassium Chloride KCl 0.300Calcium Chloride Hydrate 2X with

Water

CaCl22H2O

0.015Sodium Bicarbonate NaHCO3 2.520

Resazurin 0.050

Yeast extract 4.000

L-Cysteine 0.240

Figure A1: Basal Medium Formula


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Page A2 ofA7

Added to Completed Basal MediumAfter Autoclaving

Solution Required Milliliters (mL)

Trace Element Solution 1.00

Selenium-Tungstate solution 1.00

Vitamin Solution 5.00

Sodium Sulfide Solution 10.00

Trace Element Solution

Chemical Formula Required Milligrams (mg)

Iron Sulfate Hydrated 7X with WaterFeSO4 7H2O

2085.00

Cobalt II Chloride Hydrated 6X withWater

CoCl2 6H2O190.00

Manganese Chloride Hydrated 4Xwith Water

MnCl2 4H2O100.00

Zinc Sulfate Hydrated 7X with WaterZnSO4 7H2O

111.00

Boric Acid H3BO3 6.00Sodium Molybdate Hydrated 2X with

WaterNa2MoO4 2H2O

36.00

Nickel Chloride Hydrated 6X withWater

NiCl2 6H2O24.00

Copper II Chloride Hydrated 2X withWater CuCl2 2H2O 2.00

Figure A2: Solutions Added To Basal Medium

Figure A3: Trace Element Formula


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Page A3 ofA7

Selenium - Tungsten Solution

Solution Formula Required Milligrams (mg)

Sodium Selenite Hydrated

5X with WaterNa2SeO3 5H2O

6.00Sodium Tungstate

Hydrated 2X with WaterNa2Wo4 2H2O

8.00

Sodium Hydroxide NaOH 540.00

Vitamin Solution

Chemical Required Milligrams (mg)

Biotin 20.00

Folic Acid 20.00

Pyridoxine Hydrochloride 100.00

Riboflavin 50.00

Thiamine 50.00

Nicotinic Acid 50.00

Pantothenic Acid 50.00

Vitamin B12 1.00

p-Aminobenzoic Acid 50.00

Thioctic Acid 50.00

Figure A4: Selenium Tungsten Solution

Figure A5: Vitamin Formula


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Page A4 ofA7

Sodium Sulfide Solution

Chemical Formula Required AmountSodium Sulfide Solution

Hydrated 9 timesNa2S 9H2O

48 (mg)

Deionized Water H2O 40 (mL)

Glucose and Xylose PercentagesTreated

Sample OneTreated

Sample TwoUntreated

Sample OneUntreated

Sample Two

GlucosePercentage 48.4 49.2 33.1 33.8

XylosePercentage

17.3 19.1 16.3 13.5

AveragePercentage

Glucose48.8 33.5

AveragePercentage

Xylose18.2 14.9

Figure A6: Sodium Sulfide Solution

Figure A7: Glucose and Xylose Percentages


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Page A5 ofA7

HPLC Results Ethanol Content Ethanol Average

BacteriaEthanol, ml

ethanol per mlof solution

Ethanol,% v/v

AverageEthanol %

(a,b,c)v/v

ClostridiumThermocellum 1a 0.0720 7.20 7.18

1b 0.0545 5.45

1c 0.0890 8.90

Control 1d 0.0025 0.25Control 1e*

ClostridiumThermolactium 2a 0.0435 4.35 5.08

2b 0.0410 4.10

2c 0.0680 6.80

Control 2d 0.0012 0.12

Control 2e*

Co-culture 3a 0.1705 17.05 14.55

3b 0.1225 12.25

3c 0.1435 14.35

Control 3d 0.0210 2.10

Control 3e 0.0180 1.80* The label "*" control samples couldn't give integratable HPLC curves, probably they were toosmall, and were covered by noisy signals.

Figure A8: HPLC Results Ethanol Content


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Page A6 ofA7

TreatedSample One

TreatedSample Two

UntreatedSample One

UntreatedSample Two

Klason LigninPercentage

17.94 17.62 25.18 24.59

AveragePercentage

Klason Lignin

17.78 24.89

BacteriaTreated Average Ethanol

%(a,b,c)v/v

Untreated Average

Ethanol % (Control )

(e and d)

ClostridiumThermocellum 7.18 0.13

ClostridiumThermolactium 5.08 0.06

Co-culture 14.55 1.95

Figure A9: Klason Lignin Content

Figure A10: Average Ethanol Content


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Page A7 ofA7

Comparison of Enzymatic Hydrolysis (2012) and Bacterial Hydrolysis (2013)

Enzymatic Hydrolysis Bacterial Cellulose Hydrolysis

Biomass

(2011-2112)NaOH PretreatmentAverage Ethanol %

(a and b)v/v

(2011-2012)H2SO4 PretreatmentAverage Ethanol %

(a and b)v/v


ThermocellumAverage Ethanol %

(a,b,c)v/v


ThermolactiumAverage Ethanol %

(a,b,c)v/v

(2012-2013)Co-culture

Average Ethanol %(a,b,c)

v/v

CornStover 5.135 8.994 7.18 5.08 14.55

Figure A11: Comparison of Ethanol Content 2012 and 2013