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Slide 1

Streamlined Biofuel ProductionNext Right

Investigating the Use of Anaerobic Fermentation on Pretreated Biomass to Streamline Bio-fuel Production

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

HypothesisIf compared with last years 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

ControlsThe tools, equipment, materials, and procedures were identical within each of the three groups studied

VariablesThe two different bacteria strains and the co-culture were the 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

Corn Stover

Graphical Analysis

Grind, Detoxify and Neutralize

Acid Pretreatment No Pretreatment

EthanolRecover and Measure

Media and Autoclave

Clostridium Thermolactium

Clostridium Thermocellum

Bacterial Hydrolysis Fermentation of Sugars

Co-Culture

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 120degrees 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 Treated Sample One Two Glucose Percentage Xylose Percentage Average Percentage Glucose Average Percentage Xylose 48.4 17.3 48.8 49.2 19.1 Untreated Sample One 33.1 16.3 33.5 Untreated Sample Two 33.8 13.5

18.2

14.9

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

Slide 10

Where: mpaper+lignin mpaper msample

= = =

Oven dry weight of filter paper and lignin, mg Oven dry weight of filter paper, mg 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 Two17.62

Untreated Sample One25.18

Untreated Sample Two24.59

Percentage Average Percentage

17.94

17.78

24.89

Klason Lignin content in table

Slide 12

Basal MediumChemicalSodium Chloride Magnesium Potassium Dihydrogen Phosphate Ammonium Chloride Potassium Chloride Calcium Chloride Hydrate 2X with Water Sodium Bicarbonate Resazurin Yeast extract L-Cysteine

FormulaNaCl MgCl2.6H2O KH2PO4 NH4Cl KCl CaCl2 2H2O NaHCO3

Required Grams (g)10.000 0.500 0.200 0.300 0.300 0.015 2.520 0.050 4.000 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 milliliters 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

HPLC Results Ethanol Content Ethanol AverageBacteriaClostridium Thermocellum

Ethanol, ml ethanol per ml of solution1a 1b 1c 1d 1e* 2a 2b 2c 2d 2e* 3a 3b 3c 3d 3e 0.0720 0.0545 0.0890 0.0025

Ethanol, % v/v

Average Ethanol % (a,b,c) v/v7.18

Control Control Clostridium Thermolactium

7.20 5.45 8.90 0.25

Control Control Co-culture

0.0435 0.0410 0.0680 0.0012 0.1705 0.1225 0.1435 0.0210 0.0180

4.35 4.10 6.80 0.12 17.05 12.25 14.35 2.10 1.80

5.08

14.55

Control Control

* 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.Figure A8: HPLC Results Ethanol Content

Slide 17

16

Ethanol Percentage (v/v) ComparsionP e r c e n t a g e14

12

10

8

6

4

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.

(v / v2

)0

Clostridium Thermocellum

Clostridium Thermolactium

Co-culture

Bacterial Hydrolysis and Fermentation

Slide 18

Comparison of Enzymatic Hydrolysis (2012) and Bacterial Hydrolysis (2013) Enzymatic Hydrolysis(2011-2112) NaOH Pretreatment Biomass Average Ethanol % (a and b) v/v Corn Stover (2011-2012) H2SO4 Pretreatment Average Ethanol % (a and b) v/v

Bacterial Cellulose Hydrolysis(2012-2013) Clostridium Thermocellum Average Ethanol % (a,b,c) v/v 7.18 (2012-2013) Clostridium T