1. Presentation©E.Schmid-2011 “Biofuels Production & Analysis”

2. Overview of Biofuels Feedstocks The sun is the primary energy source for phototrophic life forms (plants, algae and cyanobacteria) which use photosynthesis to convert electromagnetic energy (E= h x v) into water-derived reduction equivalents (NADH) and ATP which with CO2 is used to build simple sugars like glucose which can be turned into: hydrocarbons: starch, cellulose, lignin and oil nucleic acids (add nitrogen and phosphorous) amino acids and proteins (add nitrogen and sulfur)

4. Photosynthesis & Its Products Atmospheric CO 2 H2OH2O Glucose/Fructose (C 6 H 12 O 6 ) Plant or Algae Sun Graphic©ElmarSchmid-2010 Solar Energy Cellulose Starch Oil Lignins O2O2 Photosynthesis Biomass

5. Photosynthesis

6. Biomass: Starch and Cellulose

7. Biomass: Lignin

8. Glucose to Phenylalanine

9. Phenylalanine to Lignin 1

10. Biomass: Oil (Triglyceride)

11. Photosynthesis

12. Presentation©E.Schmid-2011 “Biofuels Production & Analysis” Industrial Bioethanol Production = 1 st Generation Biofuel

13. Steps Of Industrial Bioethanol Production harvest feedstock (corn) mash and cook corn to release glucose ferment glucose with yeast to produce ethanol distill ethanol from mixture strain mix 15%-50% ethanol with 85% gasoline for use in automobiles

14. Industrial Bioethanol Production

15. Presentation©E.Schmid-2011 “Biofuels Production & Analysis” 2 nd Generation Biofuels

16. Use corn stover, bagasse, energy cane (high in cellulose and lignin) for feedstock It is difficult to release and ferment the sugars from these feedstocks made from cellulose and lignin that make up the plant cell wall.

17. Barriers to Cellulosic Biofuels Cellulosic Ethanol Production –The entire process is expensive –Grasses are difficult to transport and to store (corn can be stored at the farm until it is transported to the ethanol facility, grasses are bulky for storage) –Cellulosic enzymes are not as efficient as is desired* cellulose is difficult to breakdown cellulosic enzymes are expensive to produce –Efficient microbes for fermentation are still being researched –The entire process has not been optimized for commercial production *Companies such as Genencor (Danisco) In Rochester, NY and Novazyme in NC are working on the development of cellulosic enzymes. Today we will look for the cellulosic enzyme, cellobioase, in mushrooms.

18. Presentation©E.Schmid-2011 “Biofuels Production & Analysis” Hydrogen from Bacteria

19. Mira Costa College Educational biohydrogen reactor work station Photo©E.Schmid-2010

20. Bacterial Production of H 2 Fuel Prepare and sterilize media in a spinner bottle. Inoculate with Enterobacter aerogenes. Culture at room temperature until hydrogen gas is produced. Run tubing to fuel cell which strips electrons from hydrogen atoms using a platinum catalyst. Electrons pass into wire to fan, activating fan. Protons pass to other side of fuel cell and combine with oxygen to produce water.

21. Biofuels from Microalgae

22. Why Biofuels from Microalgae?

23. CropOil Yield (kg oil / ha x year) Oil Yield (gal oil / ha x year) Corn14645 Soybeans375120 Peanuts921282 Rapeseed/ Canola 1,000306 Olives1,051322 Avocado2,298705 Palm oil5,0001,575/1,890 Algae Farming 268,950 (Valcent) 60,000 (Shell) 21,842 (Molina et al.) 33,000 (other) 82,500 18,405 6,700 10,123 Comparison of Oil Production in Agricultural Plants

24. Microalgal Photosynthesis & Oils Atmospheric CO 2 H2OH2O Glucose/Fructose (C 6 H 12 O 6 ) Plant or Algae Sun Graphic©ElmarSchmid-2010 Solar Energy Cellulose Starch Oil Lignins O2O2 Photosynthesis Biomass

25.  Microalgae have a fast growth rate and can double in less than 24 hours.  Microalgae utilize the available sunlight much more efficiently than terrestrial green plants. Most microalgae have a solar conversion efficiency of about 4-5% which is higher than in plants.  Microalgae are metabolically very versatile and many value products can can be produced, including antioxidants, poly-unsaturated fatty acids, oils, and fish and cattle feed.  Large scale cultivation of microalgae removes significant amounts of the greenhouse gas CO 2 from the atmosphere, leading to net 0 CO 2 when combusted (burning fossil fuels adds CO2 to the atmosphere)  Large scale cultivation of microalgae under controlled, contamination-free conditions can be achieved in closed loop photobioreactors. Microalgae Advantages

26. Commercial Tubular Closed Loop Algae Photobioreactor Taken from the website of Bioprodukte-Prof. Steinberg GmbH, Germany

27. Bubble Column Photobioreactor Work Station Photo©E.Schmid-2010

28.  Fuels can be produced in a sustainable, renewable way - algae are harvested and quickly regrown within days or weeks within photobioreactor environments.  Fuels, e.g. biodiesel, burns carbon-neutral when combusted in internal combustion engines or other energy conversion devises.  Microalgae oils and fuels are non-toxic and highly bio-degradable.  Biodiesel is a drop-in fuel and may be used in any diesel vehicle with no engine conversion necessary. In 2012, the Navy is piloting the use of 50%/50% drop-in biofuels and fossil fuels in their vehicles in Hawaii; in 2016 the Navy will convert all its vehicles (except nuclear powered) to this mix.  Algae can grow in low grade water, waste water and even marine water. Microalgae Advantages, continued

29. Microalgae Supply Chain

30. A triglyceride is composed of one glycerol molecule chemically linked with three fatty acids Glycerol Fatty acid e.g. Stearic acid (C18:0)  Fatty acid can be saturated or unsaturated + 3x Triacyl- Glyceride (“Oil” or “Fat”) Components of Fats, Oils or Triglycerides

31. Oil Extraction Methods: Mechanical Extraction

32. Since oils are lipophilic they are often extracted from biological materials, e.g. seeds or algae, with the help of lipophilic (organic) solvents. Important organic solvents used for oil extraction are: 1. n-Hexane 2. Chloroform (CHCl 3 ) 3. Isopropanol Many different manual and automatized oil extraction methods have been developed, such as: 1. Folch method 2. Soxhlet method 3. Accelerated solvent extraction (ASE) method Oil Extraction Methods: Chemical Extraction

33. Steps of the Folch method Algae dry biomass Algae cell disintegration (Mortar, Ball milling, sonication) Chloroform/methanol (2:1) Vortex Centrifugation Chloroform transfer into new tube Chloroform (pure) Vortex Centrifugation Chloroform transfer & pooling Solvent evaporation

34. Biodiesel Biochemically, the raw material for biodiesel production are triacylglycerides (TAGs) Depending on the degree of saturation of the fatty acids, TAGs are referred to as oils or fats Biodiesel is produced via a process called transesterification Unsaturated C 16–18 Fatty Acid Methyl Esters (FAME) (“Biodiesel”) Triacylglycerides (TAGs) Transesterification using Methanol and Base (Methoxide) Oils Fats

35. Biodiesel

37. Biodiesel Analysis Fatty acid methyl esters (FAMEs) can be detected and analyzed by different methods including: 1. Gas chromatography (GC) 2. High pressure liquid chromatography (HPLC) Gas chromatography is highly sensitive but requires prior derivatization of FAME sample HPLC-based analysis requires special detection system called evaporative light scattering detection (ELSD) Both methods give typical product peaks of the analyzed FAME sample

38. Typical Result Of FAME Analysis With HPLC-ELSD 13 min0 min Yielded information: 1. Retention time 2. Area under the curve (peak)  quantity

39. Another Alternative? Pyrolysis of Duckweed http://www.youtube.com/watch?v=4bJVvEd -cRk