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Similar to Anaerobic Fermentation Streamlines Biofuel Production
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Anaerobic Fermentation Streamlines Biofuel Production
- 1. Investigating the Use of Anaerobic Fermentation on Pretreated Biomass to Streamline Bio-fuel Production Streamlined Biofuel Production Next Right
- 2. Contents Hypothesis Controls - Variables Bacteria Studied Methods and Materials Results Acknowledgements
- 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.
- 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
- 5. Bacteria Studied Clostridium Thermocellum Clostridium Thermolactium Co-Culture
- 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
- 9. Glucose and Xylose Percentages Treated Sample One Treated Sample Two Untreated Sample One Untreated Sample Two Glucose Percentage 48.4 49.2 33.1 33.8 Xylose Percentage 17.3 19.1 16.3 13.5 Average Percentage Glucose 48.8 33.5 Average Percentage Xylose 18.2 14.9
- 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
- 11. Klason Lignin Content Treated Sample One Treated Sample Two Untreated Sample One Untreated Sample Two Percentage 17.94 17.62 25.18 24.59 Average Percentage 17.78 24.89
- 12. 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 0.300 Calcium Chloride Hydrate 2X with Water CaCl2 2H2O 0.015 Sodium Bicarbonate NaHCO3 2.520 Resazurin 0.050 Yeast extract 4.000 L-Cysteine 0.240
- 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
- 16. 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.
- 17. 0 2 4 6 8 10 12 14 16 Clostridium Thermocellum Clostridium Thermolactium Co-culture P e r c e n t a g e ( v / v ) Bacterial Hydrolysis and Fermentation Ethanol Percentage (v/v) Comparsion
- 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
- 19. 0 2 4 6 8 10 12 14 16 NaOH Pretreatment H2SO4 Pretreatment Clostridium Thermocellum Clostridium Thermolactium 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
- 20. Acknowledgments 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.
Editor's Notes
- 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.