by daniel c. perrinez morse hall 103 faculty contact: ihab h. … · 2015. 9. 18. · 0.079...
TRANSCRIPT
By Daniel C. Perrinez
Morse Hall 103
Faculty Contact: Ihab H. Farag, Sc.D., P.E.
Overview
Background
Project
Process Description
Approach
Chemicals, Equipment , and Wastes
Status & Future Steps
http://www.rbgsyd.nsw.gov.au/__data/assets/image/0019/47350/chlorella2.gif
Picture of Triglyceride Lipid
Transesterfication
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.
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)
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)
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)
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)
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
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
~2mm @ 1000x
Approach
Cell Disruption Experiments
Experiment 1:
Absorbance Method
0.079 Blank/Unbroken Cells
0.175 Sonication ~20sec
0.185 Sonication + an extra ~20sec
0.170 Sonication + ½ volume .3-.5mm glass beads ~ 20 sec
0.135 Nitrogen gas press @ 170 psi
0.120 Glass tissue grinder~ 1min
0.150 Glass tissue grinder + ½ volume .3-.5mm glass beads ~ 1min
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)
Approach
Lipid Extraction:
Cell Disruption
1. Homogenize (10 hr)
1. Sonicate (15 min)
1. Teflon Grinder (10 min)
1. Homogenize (5 hr)
Approach
Lipid Extraction:
Cell Disruption
1. Homogenize (10 hr)
1. Sonicate (15 min)
1. Teflon Grinder (10 min)
1. Homogenize (5 hr)
Approach
Lipid Extraction:
Cell Disruption
1. Homogenize (10 hr)
1. Sonicate (15 min)
1. Teflon Grinder (10 min)
1. Homogenize (5 hr)
Approach
Lipid Extraction:
Cell Disruption
1. Homogenize (10 hr)
1. Sonicate (15 min)
1. Teflon Grinder (10 min)
1. Homogenize (5 hr)
Approach
Lipid Extraction:5. Centrifuge
6. Evaporate off solvent
(Chloroform: Methanol)
7. Preserve
○ Freezer (0˚C)
○ Nitrogen atmosphere
○ Butylated hydroxytoluene (BHT)
Approach
Lipid Analysis: HPLC
○ Phospholipids
○ Glycolipids
GC
○ Fatty Acid Methyl Esters (FAMEs)
Gas Chromatography
FAMEs
were
detected
But, did not
separate
well
May be an
old column
Matrix Assisted Laser
Desorption/ Ionization
Mass measurements
are essential to
confirming HPLC and
GC readings.
matrix of 2,5
dihydroxybenzoic acid
C18:3 standard
C19 standard
C16 standard
C19:H1A (30:70; in-situ
transesterfication
Chemicals, Equipment , and Wastes
Chemicals used: Function:
Chloroform (CHCl3) Solvent
Methanol (CH3OH) Solvent
Butylated hydroxytoluene (C15H24O) Antioxidant
Sedmax’s Solution Protein binding dye
Nitrogen Gas Inert atmosphere
Nile Red Lipid fluorescent dye
Phosphate Buffer 2/10mol (K2HPO4) Ph buffer solution for Sedmax test
2,5-dihydroxybenzoic acid MALDI matrix
Chemicals, Equipment , and Wastes
Equipment used: For use in:
Spectrophotometer Sedmax test
Spectroflourometer Nile red test
GC + columns Lipid separation + quantification
Teflon Grinder Cell disruption
Sonicator Cell disruption
Nitrogen gas tank & two-stage regulator
Lipid Extraction
Centrifuge (large and small sizes)
Lipid Extraction
Freeze dryer Preparation
MALDI Mass Measurement
Chemicals, Equipment , and Wastes
Waste byproducts:
Algae biomass
Chloroform (CHCl3)
Methanol (CH3OH)
Sedmax’s solution
Analyzed lipids
Water
Nile Red solution
Phosphate Buffer 2/10mol (K2HPO4)
Acknowlegements
Dr. Ihab H. Farag
Professor of Chemical Engineering
W315 Kingsbury Hall
(603) 862-2313
Dr. Leland S. Jahnke
Professor of Plant Biology
Rudman Hall Rm 183
(603) 862-3857
Nancy L.Whitehouse (Agricultural Experiment Stat)
Dr. Samuel C. Smith
Professor of Emeritus (Active)-Molecular,
Cellular, Biomed SC
Kendall Hall Rm 13
(603) 862-2505
John E. Newell
ENGINEERING TECHNICIAN
Kingsbury Hall
(603) 86 2-1921
References
Briggs, Widescale Biodiesel Production from Algae [Internet]: University of New Hampshire, Physics Department; c2004 [cited 2009 9/4]. Available from:
http://www.unh.edu/p2/biodiesel/article_alge.html .
Erwin JA, editor. 1973. Lipids and biomembranes of eukaryotic microorganisms. New York: Academic Press.
Ferrentino J. 2007. Microalgal oil extraction and in situ transesterification.
Folch J, Lees M, Sloane-Stanley G. 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. (226):497-509.
Gerpen JV, Shanks B, Pruszko R. 2004. Biodiesel production technology .
Guckert JB, Cooksey KE, Jackson LL. 1988. Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga, chlorella. J
Microbiol Methods 8(3):139-49.
Gurr MI, Harwood JL, Frayn KN. 2002. Lipid biochemistry: An introduction. 5th ed. Oxford, England: Blackwell Science.
Sheehan J, Dunahay T, Benemann J, Roessler P. 1998. 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 80401-3393.
Schiller J, Arnhold J, Benard S, Müller M, Reichl S, Arnold K. 1999. Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A
methodological approach. Anal Biochem 267(1):46-56.
Questions?