By Daniel C. Perrinez Morse Hall 103. Faculty Contact: Ihab H. Farag, Sc.D., P.E.

Transcription

1 By Daniel C. Perrinez Morse Hall 103 Faculty Contact: Ihab H. Farag, Sc.D., P.E.

2 Overview Background Project Process Description Approach Chemicals, Equipment, and Wastes Status & Future Steps

3 data/assets/image/0019/47350/chlorella2.gif

4 Picture of Triglyceride Lipid

5 Transesterfication

6

7 Project Objectives: Develop lipid extraction for Chlorella sp. (micro-algae) Develop Lipid profiling method for Chlorella sp.: Identify Lipids by class Quantify Lipid class constituents Goal: Observe trends between the abundance of triglyceride molecules and other in-vivo lipids.

8 Process Description Microalgae are grown in a photo-bioreactor Cells are lypholized Lipids are extracted from Cells Lipid molecules are separated & quantified by High-performance liquid chromatography (HPLC) Gas Chromatography (GC)

9 Process Description Microalgae are grown in bio reactor Cells are lypholized (freeze dried) Lipids are extracted from Cells Lipid molecules are separated & quantified by High-performance liquid chromatography (HPLC) Gas Chromatography (GC)

10 Process Description Microalgae are grown in bio reactor Cells are lypholized Lipids are extracted from Cells Lipid molecules are separated & quantified by High-performance liquid chromatography (HPLC) Gas Chromatography (GC)

11 Process Description Microalgae are grown in bio reactor Cells are lypholized Lipids are extracted from Cells Lipid molecules are separated & quantified by: Gas Chromatography (GC) High-performance liquid chromatography (HPLC)

12 Approach Lots of Reading! Difficulties: Chlorella is Small! (~2-10μm) and hard to break Limited prior research is speculative Not all lipids can dissolve in just one solvent

13 Approach Lots of Reading! Difficulties: Chlorella is Small! (~2-10μm) and hard to break Limited prior research is speculative 1000x Not all lipids can dissolve in just one solvent

14 Approach Cell Disruption Experiments Experiment 1: Absorbance Method Blank/Unbroken Cells Sonication ~20sec Sonication + an extra ~20sec Sonication + ½ volume.3-.5mm glass beads ~ 20 sec Nitrogen gas 170 psi Glass tissue grinder~ 1min Glass tissue grinder + ½ volume.3-.5mm glass beads ~ 1min

15 Approach Cell Disruption Experiments Experiment 2 Absorbance Method 0.36 Blank/Unbroken Cells 0.41 Mortar & Pestle (5min) 0.46 Teflon grinder (5min) 0.45 Sonication (40 sec) + Mortar & Pestle (5min) 0.49 Sonication (40 sec) + Teflon grinder (5min) 0.41 Sonication (40 sec)

16 Approach Lipid Extraction: Cell Disruption 1. Homogenize (10 hr) 1. Sonicate (15 min) 1. Teflon Grinder (10 min) 1. Homogenize (5 hr)

17 Approach Lipid Extraction: Cell Disruption 1. Homogenize (10 hr) 1. Sonicate (15 min) 1. Teflon Grinder (10 min) 1. Homogenize (5 hr)

18 Approach Lipid Extraction: Cell Disruption 1. Homogenize (10 hr) 1. Sonicate (15 min) 1. Teflon Grinder (10 min) 1. Homogenize (5 hr)

19 Approach Lipid Extraction: Cell Disruption 1. Homogenize (10 hr) 1. Sonicate (15 min) 1. Teflon Grinder (10 min) 1. Homogenize (5 hr)

20 Approach Lipid Extraction: 5. Centrifuge 6. Evaporate off solvent (Chloroform: Methanol) 7. Preserve Freezer (0 C) Nitrogen atmosphere Butylated hydroxytoluene (BHT)

21 Approach Lipid Analysis: HPLC Phospholipids Glycolipids GC Fatty Acid Methyl Esters (FAMEs)

22 Gas Chromatography FAMEs were detected But, did not separate well May be an old column

23 Matrix Assisted Laser Desorption/ Ionization Mass measurements are essential to confirming HPLC and GC readings. matrix of 2,5 dihydroxybenzoic acid

24 C19 standard C18:3 standard C16 standard C19:H1A (30:70; in-situ transesterfication

25 Chemicals, Equipment, and Wastes Chemicals used: Chloroform (CHCl 3 ) Methanol (CH 3 OH) Butylated hydroxytoluene (C 15 H 24 O) Function: Solvent Solvent Antioxidant Sedmax s Solution Nitrogen Gas Nile Red Phosphate Buffer 2/10mol (K 2 HPO 4 ) Protein binding dye Inert atmosphere Lipid fluorescent dye Ph buffer solution for Sedmax test 2,5-dihydroxybenzoic acid MALDI matrix

26 Chemicals, Equipment, and Wastes Equipment used: Spectrophotometer Spectroflourometer For use in: Sedmax test Nile red test GC + columns Lipid separation + quantification Teflon Grinder Sonicator Nitrogen gas tank & twostage regulator Centrifuge (large and small sizes) Freeze dryer MALDI Cell disruption Cell disruption Lipid Extraction Lipid Extraction Preparation Mass Measurement

27 Chemicals, Equipment, and Wastes Waste byproducts: Algae biomass Chloroform (CHCl 3 ) Methanol (CH 3 OH) Sedmax s solution Analyzed lipids Water Nile Red solution Phosphate Buffer 2/10mol (K 2 HPO 4 )

28 Acknowlegements Dr. Ihab H. Farag Professor of Chemical Engineering W315 Kingsbury Hall (603) Dr. Leland S. Jahnke Professor of Plant Biology Rudman Hall Rm 183 (603) Dr. Samuel C. Smith Professor of Emeritus (Active)-Molecular, Cellular, Biomed SC Kendall Hall Rm 13 (603) John E. Newell ENGINEERING TECHNICIAN Kingsbury Hall (603) Nancy L.Whitehouse (Agricultural Experiment Stat)

29 References Briggs, Widescale Biodiesel Production from Algae [Internet]: University of New Hampshire, Physics Department; c2004 [cited /4]. Available from: Erwin JA, editor Lipids and biomembranes of eukaryotic microorganisms. New York: Academic Press. Ferrentino J Microalgal oil extraction and in situ transesterification. Folch J, Lees M, Sloane-Stanley G A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. (226): Gerpen JV, Shanks B, Pruszko R Biodiesel production technology. Guckert JB, Cooksey KE, Jackson LL Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga, chlorella. J Microbiol Methods 8(3): Gurr MI, Harwood JL, Frayn KN Lipid biochemistry: An introduction. 5th ed. Oxford, England: Blackwell Science. Sheehan J, Dunahay T, Benemann J, Roessler P A look back at the U.S. department of Energy s aquatic species Program bio-diesel from algae. U.S. Department of Energy s Office of Fuels Development, National Renewable Energy Laboratory 1617 Cole Boulevard. Golden, Colorado Schiller J, Arnhold J, Benard S, Müller M, Reichl S, Arnold K Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A methodological approach. Anal Biochem 267(1):46-56.

30 Questions?