Slide 1

Investigating the Use of Anaerobic Fermentation on

Pretreated Biomass to Streamline Bio-fuel Production

Streamlined

Biofuel Production

Next Right

Title Page The essential purpose of this research was to discover a new approach to realize a superior yield when producing Cellulosic ethanol. Cellulosic biofuel is attractive because, rather than utilize food crops, such as corn, it transforms farm waste into a renewable and clean-burning biofuel. The current method using enzymatic hydrolysis to release sugars followed by a separate yeast fermentation has made biofuel production profitable and reduced gas prices at the pump by as much as $1.09 in 2012. However, this requires a two-step process and can only effectively ferment glucose, whereas bacterial hydrolysis releases both glucose and xylose from the biomass and ferments it all in one step. By using two bacteria strains that efficiently degrade different sugars, I thought to increase the ethanol yield, and this proved to be true. The co-culture that I evaluated for this study dramatically increased ethanol yield.

Slide 2

Contents

Hypothesis

Controls - Variables

Bacteria Studied

Methods and Materials

Results

Acknowledgements

References

Here are the things we will be covering. Slide 3

Hypothesis

If compared with last year’s study of

enzymatic hydrolysis, single-strain

bacterial cellulose hydrolysis will be

proven to produce more ethanol;

whereas combining two strains of

bacteria in a co-culture will yield the

highest percentage of ethanol.

Hypothesis I believed that bacteria would produce more ethanol than enzymes and that the co-culture, using equal portions of the two bacteria, would be the most efficient of the processes. This was based on information about the two bacteria strains that were used in this study. The first, Clostridium Thermocellum efficiently degrades hexoses, monosaccharides with six carbon atoms, while the second, Clostridium thermolactium proficiently degrades pentoses, monosaccharides with five carbon atoms. Therefore, I expected the co-culture to convert most of the sugars and produce the highest percentage of ethanol.

Slide 4

Controls

The tools, equipment, materials, and

procedures were identical within each

of the three groups studied

The two different bacteria strains and

the co-culture were the variables

Variables

Controls and Variables These things were all identical in each experiment. The bacteria strains were the variables Slide 5

Bacteria Studied

Clostridium Thermocellum

Clostridium Thermolactium

Co-Culture

Bacteria Studied Here you see the three bacteria strains I tested The co-culture contained equal part of Clostridium Thermocellum and Clostridium thermolactium

Slide 6

Collect and

Dry Materials

Grind, Detoxify

and Neutralize

Recover and

Measure

Ethanol

Graphical

Analysis

Bacterial Hydrolysis

Fermentation of

Sugars

No

Pretreatment

Acid

Pretreatment

Co-Culture

Media and

Autoclave

Clostridium

Thermolactium

Clostridium

Thermocellum

Corn Stover

Project Flow Chart Here is a flowchart showing the process I followed. Slide 7

Grinding Biomass and Acid Pretreatment The biomass was cut, dehydrated, and then ground to the size of 50-micron particles After pretreating the biomass with a 1% sulfuric acid solution under pressure and at 120-degrees Celsius to break down the lignin and release the sugars, I washed it to a pH of seven.

Slide 8

Determination of Klason Lignin Here is the filtrate in the back with the Klason Lignin on the filters in front. Slide 9

Glucose and Xylose PercentagesTreated Sample

One

Treated Sample

Two

Untreated

Sample One

Untreated

Sample Two

Glucose

Percentage48.4 49.2 33.1 33.8

Xylose

Percentage17.3 19.1 16.3 13.5

Average

Percentage

Glucose

48.8 33.5

Average

Percentage

Xylose

18.2 14.9

Sugar Content To determine the percentages of glucose and xylose, I analyzed the filtrate using high-performance liquid chromatography - you see the results here

Slide 10

Where: mpaper+lignin = Oven dry weight of filter paper and lignin, mg mpaper = Oven dry weight of filter paper, mg msample = Oven dry weight of sample, mg

Formula to Determine Klason Content The Klason Lignin content was determined using this formula. Slide 11

Klason Lignin ContentTreated Sample

One

Treated Sample

Two

Untreated Sample

One

Untreated Sample

Two

Percentage 17.94 17.62 25.18 24.59

Average

Percentage17.78 24.89

Klason Lignin content in table

Slide 12

Basal MediumChemical Formula Required Grams (g)

Sodium Chloride NaCl 10.000

Magnesium MgCl2.6H2O 0.500

Potassium Dihydrogen

PhosphateKH2PO4 0.200

Ammonium Chloride NH4Cl 0.300

Potassium Chloride KCl 0.300

Calcium Chloride Hydrate

2X with WaterCaCl2 2H2O 0.015

Sodium Bicarbonate NaHCO3 2.520

Resazurin 0.050

Yeast extract 4.000

L-Cysteine 0.240

Basal Media All of the processes undertaken up to this point have been to analyze the biomass. Understanding the composition of the biomass is vital to designing the perfect method of pretreatment, hydrolysis and fermentation that will lead to production of the highest ethanol yield in the most cost-effective and sustainable system. Now I was able to begin the bacterial hydrolysis process. First, I created the basal medium using this list of components Slide 13

Introducing Bacteria into Media Utilizing a precise scale, I isolated fifteen, .002-gram specimens of biomass and placed them in 15, ten-mL, sterile serum bottles, I added five milliliter’s of the basal media to each bottle and boiled them to remove the oxygen – point to resazurin – the oxygen indicator, Resazurin, in the media changes from a rose color to very pale yellow when the oxygen has been depleted from the media Next, I autoclaved the samples for 20 minutes at 20 pounds per square inch and 121 degrees Celsius to remove all undesirable organisms My next step was to create the perfect environment for the bacteria to thrive by adding the following four solutions - Point to table of added solutions - Trace Element Solution Selenium - Tungstate solution Vitamin Solution Sodium Sulfide Solution-to remove the remaining oxygen Now I was ready to introduce the bacteria – utilizing a hypodermic syringe, I added .5 ml of Clostridium thermocellum to five samples, .5 ml of Clostridium thermolactium to another five samples, and .25 ml of each (creating a co-culture)to the last five

Slide 14

Incubating Bacteria and Biomass Then I placed the bottles in the incubator at 60 degrees Celsius for five days to create the optimal environment to facilitate effective bacterial hydrolysis and fermentation of the sugars The last step was determining ethanol percentages through use of the High-Performance Liquid Chromatography (HPLC) To do this, I calibrated the HPLC by running pure ` samples of ethanol The biomass samples were then filtered and placed in the HPLC at specific locations that identified them by material and as “a”, “b”, or “c” “d” or “e” samples; and processed to determine the ethanol content. This was a long process, taking about sixty hours to complete each test.

Slide 15

Results

Based on high ethanol content, it

was concluded that the most viable

choice for large-scale production

was the co-culture

Clostridium Thermocellum produced

more ethanol than Clostridium

Thermolactium in the single-strain

trials

After examining the results of the bacteria strains tested, it was concluded that the most advantageous bacteria choice for large-scale ethanol production was the co-culture. This was based on high ethanol content. Slide 16

Figure A8: HPLC Results Ethanol Content

HPLC Results – Ethanol Content – Ethanol Average

Bacteria

Ethanol, ml ethanol per ml

of solution

Ethanol, % v/v

Average Ethanol %

(a,b,c) v/v

Clostridium Thermocellum 1a 0.0720 7.20 7.18

1b 0.0545 5.45

1c 0.0890 8.90

Control 1d 0.0025 0.25

Control 1e*

Clostridium Thermolactium 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 too small, and were covered by noisy signals.

Figure A8: HPLC Results Ethanol Content

Results Table and Graph The five samples of the Clostridium thermolactium strain performed poorly, producing less ethanol than either the Clostridium thermocellum or the co-culture. However, when the two strains were combined in the co-culture, there was a dramatic increase in fermented sugars that surpassed any method previously tested.

Slide 17

0

2

4

6

8

10

12

14

16

ClostridiumThermocellum

ClostridiumThermolactium

Co-culture

P

e

r

c

e

n

t

a

g

e

(

v

/

v)

Bacterial Hydrolysis and Fermentation

Ethanol Percentage (v/v) Comparsion

Chart Showing Ethanol Percentages – Bacterial Hydrolysis and Fermentation So, after analyzing the test results, it was clear that anaerobic fermentation using a co-culture is a feasible method of producing ethanol. Slide 18

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

Enzymatic Hydrolysis Bacterial Cellulose Hydrolysis

Biomass

(2011-2112) NaOH Pretreatment Average Ethanol %

(a and b) v/v

(2011-2012) H2SO4 Pretreatment Average Ethanol %

(a and b) v/v

(2012-2013) Clostridium

Thermocellum Average Ethanol %

(a,b,c) v/v

(2012-2013) Clostridium

Thermolactium Average Ethanol %

(a,b,c) v/v

(2012-2013) Co-culture

Average Ethanol % (a,b,c)

v/v

Corn Stover 5.135 8.994 7.18 5.08

14.55

Figure A11: Comparison of Ethanol Content 2012 and 2013

Comparison of Ethanol Content 2012 and 2013

Comparison of Enzymatic Hydrolysis 2012 and Bacterial Hydrolysis 2013 Here is a comparison of last year’s trials with enzymatic hydrolysis with its separate yeast fermentation and this year’s study of the one-step bacterial hydrolysis and fermentation.

Slide 19

Figure A8: HPLC Results Ethanol Content

0

2

4

6

8

10

12

14

16

NaOHPretreatment

H2SO4Pretreatment

ClostridiumThermocellum

ClostridiumThermolactium

Co-culture

P

e

r

c

e

n

t

a

g

e

(

v

/

v)

Ethanol Percentage (v/v) Comparsion 2012-2013

Enzymatic Hydrolysis and Yeast Fermentation – 2012

Bacterial Hydrolysis and Fermentation – 2013

The comparison illustrated in a bar graph. Slide 20

Acknowledgments

I thank Dr. Ulrike Tschirner, from theUniversity of Minnesota, for her generousand steadfast assistance with equipmentand materials, with bacteria, and withinformed counsel.

I 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. Slide 21

References• Energy Efficiency and Renewable Energy, 2011, Crop Residues and Agricultural Wastes

http://www1.eere.energy.gov/biomass/pdfs/btu_crop_residues.pdf

• Osborne, Stefan, 2007, Energy in 2020: Assessing the Economic Effects of Commercialization of Cellulosic Ethanol

http://www.trade.gov/mas/ian/build/groups/public/@tg_ian/documents/webcontent/tg_ian_002699.pdf

• Milbrandt, Anelia, 2006, Geographic Perspective on the Current Biomass Resource Availability in the United States, NREL

http://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 Terms

http://www.nrel.gov/biomass/glossary.html

• Lane, Jim, 2010, EPA Confirms Tiny Cellulosic Biofuels Mandate for 2011

http://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 More than $1 in 2011

http://www.ethanolrfa.org/

• Urbanchuk, John, 2010, Current State of the U.S Ethanol Industry, Cardno Entrix

http://www.nrel.gov/analysis/pdfs/doe-02-5025.pdf

• Zverlov, Vladimir, Schwarz, Wolfgang, 2008. Bacterial Cellulose Hydrolysis in Anaerobic Environmental Subsystems—Clostridium thermocellum and Clostridium stercorarium, Thermophilic Plant-fiber Degraders. Annals of the New York Academy of Science March 2008.

http://onlinelibrary.wiley.com/doi/10.1196/annals.1419.008/abstract

• National Renewable Energy Laboratory. Capabilities in Biomass Process and Sustainability Analyses

http://www.nrel.gov/biomass/pdfs/40742.pdf