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  • Title PageThe 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.
  • Here are the things we will be covering.
  • HypothesisI 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.
  • Controls and VariablesThese things were all identical in each experiment.The bacteria strains were the variables
  • Bacteria StudiedHere you see the three bacteria strains I testedThe co-culture contained equal part of Clostridium Thermocellum and Clostridium thermolactium
  • Project Flow ChartHere is a flowchart showing the process I followed.
  • Grinding Biomass and Acid PretreatmentThe biomass was cut, dehydrated, and then ground to the size of 50-micron particlesAfter 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.
  • Determination of Klason LigninHere is the filtrate in the back with the Klason Lignin on the filters in front.
  • Sugar ContentTo determine the percentages of glucose and xylose, I analyzed the filtrate using high-performance liquid chromatography - you see the results here
  • Formula to Determine Klason ContentThe Klason Lignin content was determined using this formula.
  • Klason Lignin content in table
  • Basal MediaAll 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
  • Introducing Bacteria into MediaUtilizing 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 organismsMy 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 SolutionSelenium - Tungstate solutionVitamin SolutionSodium 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
  • 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 ethanolThe 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.
  • 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.
  • Results Table and GraphThe 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.
  • Chart Showing Ethanol Percentages – Bacterial Hydrolysis and FermentationSo, after analyzing the test results, it was clear that anaerobic fermentation using a co-culture is a feasible method of producing ethanol.
  • Comparison of Enzymatic Hydrolysis 2012 and Bacterial Hydrolysis 2013Here 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.
  • The comparison illustrated in a bar graph.
  • 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.  
  • Transcript

    • 1. Investigating the Use of Anaerobic Fermentation onPretreated Biomass to Streamline Bio-fuel ProductionStreamlinedBiofuel ProductionNext Right
    • 2. Contents Hypothesis Controls - Variables Bacteria Studied Methods and Materials Results Acknowledgements
    • 3. HypothesisIf compared with last year’s study ofenzymatic hydrolysis, single-strainbacterial cellulose hydrolysis will beproven to produce more ethanol;whereas combining two strains ofbacteria in a co-culture will yield thehighest percentage of ethanol.
    • 4. ControlsThe tools, equipment, materials, andprocedures were identical within eachof the three groups studiedThe two different bacteria strains andthe co-culture were the variablesVariables
    • 5. Bacteria StudiedClostridium ThermocellumClostridium ThermolactiumCo-Culture
    • 6. Collect andDry MaterialsGrind, Detoxifyand NeutralizeRecover andMeasureEthanolGraphicalAnalysisBacterial HydrolysisFermentation ofSugarsNoPretreatmentAcidPretreatmentCo-CultureMedia andAutoclaveClostridiumThermolactiumClostridiumThermocellumCorn Stover
    • 7. Glucose and Xylose PercentagesTreated SampleOneTreated SampleTwoUntreatedSample OneUntreatedSample TwoGlucosePercentage48.4 49.2 33.1 33.8XylosePercentage17.3 19.1 16.3 13.5AveragePercentageGlucose48.8 33.5AveragePercentageXylose18.2 14.9
    • 8. Where:mpaper+lignin = Oven dry weight of filter paper and lignin, mgmpaper = Oven dry weight of filter paper, mgmsample = Oven dry weight of sample, mg
    • 9. Klason Lignin ContentTreatedSampleOneTreatedSampleTwoUntreatedSampleOneUntreatedSampleTwoPercentage 17.94 17.62 25.18 24.59AveragePercentage17.78 24.89
    • 10. Basal MediumChemical Formula Required Grams (g)Sodium Chloride NaCl 10.000Magnesium MgCl2.6H2O 0.500Potassium DihydrogenPhosphateKH2PO40.200Ammonium Chloride NH4Cl 0.300Potassium Chloride KCl 0.300Calcium Chloride Hydrate2X with WaterCaCl2 2H2O0.015Sodium Bicarbonate NaHCO3 2.520Resazurin 0.050Yeast extract 4.000L-Cysteine 0.240
    • 11. Results Based on high ethanol content, itwas concluded that the most viablechoice for large-scale productionwas the co-culture Clostridium Thermocellum producedmore ethanol than ClostridiumThermolactium in the single-straintrials
    • 12. HPLC Results – Ethanol Content – Ethanol AverageBacteriaEthanol, mlethanol per mlof solutionEthanol,% v/vAverageEthanol %(a,b,c)v/vClostridiumThermocellum 1a 0.0720 7.20 7.181b 0.0545 5.451c 0.0890 8.90Control 1d 0.0025 0.25Control 1e*ClostridiumThermolactium 2a 0.0435 4.35 5.082b 0.0410 4.102c 0.0680 6.80Control 2d 0.0012 0.12Control 2e*Co-culture 3a 0.1705 17.05 14.553b 0.1225 12.253c 0.1435 14.35Control 3d 0.0210 2.10Control 3e 0.0180 1.80* The label "*" control samples couldnt give integratable HPLC curves, probably they were toosmall, and were covered by noisy signals.
    • 13. 0246810121416ClostridiumThermocellumClostridiumThermolactiumCo-culturePercentage(v/v)Bacterial Hydrolysis and FermentationEthanol Percentage (v/v) Comparsion
    • 14. Comparison of Enzymatic Hydrolysis (2012) and Bacterial Hydrolysis (2013)Enzymatic Hydrolysis Bacterial Cellulose HydrolysisBiomass(2011-2112)NaOH PretreatmentAverage Ethanol %(a and b)v/v(2011-2012)H2SO4 PretreatmentAverage Ethanol %(a and b)v/v(2012-2013)ClostridiumThermocellumAverage Ethanol %(a,b,c)v/v(2012-2013)ClostridiumThermolactiumAverage Ethanol %(a,b,c)v/v(2012-2013)Co-cultureAverage Ethanol %(a,b,c)v/vCornStover 5.135 8.994 7.18 5.08 14.55Figure A11: Comparison of Ethanol Content 2012 and 2013Comparison of Ethanol Content 2012 and 2013
    • 15. 0246810121416NaOHPretreatmentH2SO4PretreatmentClostridiumThermocellumClostridiumThermolactiumCo-culturePercentage(v/v)Ethanol Percentage (v/v) Comparsion 2012-2013Enzymatic Hydrolysis and YeastFermentation – 2012Bacterial Hydrolysis andFermentation – 2013
    • 16. AcknowledgmentsI thank Dr. Ulrike Tschirner, from theUniversity of Minnesota, for her generousand steadfast assistance with equipmentand materials, with bacteria, and withinformed counsel.