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Standard Measurement Technologies
Nile Red is the current measurement tool of choice in the algae industry
Here’s what Nile red measures: lipophilic molecules
Here’s an example of how Nile red measurements can mislead algae growers:
Nile red measurements may indicate that both of these samples has 30% “oil”
UT/OpenAlgae Technologies
Microscopy Spectroscopy Chromatography/Mass Spectrometry
1. Scanning Electron Microscopy (SEM)
1. UV-Vis Spectrophotometry 1. Thin Layer Chromatography (TLC)
2. Transmission Electron Microscopy (TEM)
2. Nuclear Magnetic Resonance (NMR)
2. High Performance Liquid Chromatography (HPLC/MS)
3. Light Microscopy (LM) 3. Raman 3. Gas Chromatography (GC/MS)
4. Infrared (IR)
Evaluating the intermediate products requires many analytical tools
UT/OpenAlgae Technologies – Thin Layer Chromatography
Samples are taken before and after each processing step. Each sample is separated into a biomass pellet (P) and supernatant (S) fraction and analyzed by TLC against known standards.
UT/OpenAlgae Technologies – Thin Layer Chromatography
• Triglycerides remain relatively stable throughout processing, whereas diglycerides and free fatty acids rise during processing
• Suggests that we’re recovering beneficial lipids from sources other than triglycerides…possibly membrane lipids
Lipids released into the supernatantLipids remaining with the biomass pellet
UT/OpenAlgae Technologies – HPLC/MS
Quantitative Chemical Analysis of Oil Extraction ProcessHPLC methods have been developed for the quantification of algal lipid classes using Evaporative Light Scattering Detection (ELSD) and Mass Spectrometry (MS)
HC
BC
TAG
DAG
MAG FFA
Chl
Polar Lipids
More than 100 discrete ion species have been observed in lipid extracts using MS
Hydrocarbons (HC)Prenol lipids (e.g.; β-carotene, BC)Triacylglycerides (TAG)Diacylglycerides (DAG)Monoacylglyceride (MAG)Free Fatty Acids (FFA)Polar Lipids (i.e.; phospholipids)
Glycolipids
polar lipidsneutral lipids
CEM Chlorella sp.
UT/OpenAlgae Technologies – GC/MS
We can also track specific fatty acids, such as Omega 3-6-7-9, throughout growth and processing
GC/MS(gas chromatography)
• GC – identify lipid species by chain length Peak identification in GC profile above: (1) caprylic acid (C8:0); (2) capric acid (C10:0); (3) lauric acid (C12:0); (4) myristoleic acid (C14:1); (5) myristic acid (C14:0); (6) pentadecanoic acid (C15:0); (7) palmitoleic acid (C16:1); (8) palmitic acid (C16:0); (9) heptadecanoic acid (C17:0); (10) linoleic acid (C18:2n-6c); (11) oleic acid (C18:1n-9c); (12) -linolenic acid (C18:3n-3); (13) stearic acid (C18:0); (14) arachidonic acid (C20:4n-6); (15) eicosapentaenoic acid (C20:5n-3); (16) eicosenoic acid (C20:1); (17) arachidic acid (C20:0); (18) docosahexaenoic acid (C22:6n-3); (19) erucic acid (C22:1); (20) behenic acid (C22:0); (21) nervonic acid (C24:1); (22) hexacosanoic acid (C26:0); (23) octacosanoic acid (C28:0); (I.S.) tricosanoic acid (C23:0).
UT/OpenAlgae Technologies – Analysis Conclusions
• The algae industry has relied on crude quantitative technologies that can mislead growers, researchers, and investors
• UT sets itself apart by incorporating cutting edge technologies that measure lipids individually
• Adoption of these technologies will help increase understanding of algal lipid dynamics and standardize the way lipids are measured in the industry.
The Diversity of Algae
• There are ~100,000 species of algae
• Certain kinds of algae make products that we’re interested in
• oil for fuel
• high in proteins and/or carbohydrates (animal or aquaculture feeds)
• agar (thickener)
• carrageenan (stabilizer/emulsifier)
• nutraceuticals (carotenoids, omega 3-6-7-9, anti-microbials, anti-fungals)
Omega Oils – Omega 7
The problem: Limited vascularization at the wound site
A Possible Solution: Omega-7
The essential fatty acid palmitoleic acid (Omega-7) promotes the formation of new blood vessels and collagen deposition at the site of injury.
• Currently, Omega-7 is primarily derived from Sea Buckthorn, a cold weather plant that is harvested once a year.
• We have identified an algae that produces large quantities of Omega-7 and can be harvested daily.
Omega Oils – Commercially Available Omega 7 Wound Healing Study
Preliminary Results
Omega-7 accelerates wound closure
Wound Closure
Omega Oils – Commercially Available Omega 7 Wound Healing Study
Preliminary Results
Omega-7 extracts improve blood flow and minimizes scar formation in wounded sheep.
Blood Flow Due to NeovascularizationScar Formation
Omega 7 – A Collaborative Approach
Algae Scale-up and Processing In Vitro and in Vivo Study
The results of the study will be published, and if successful, be extended to industry.
Algae Biomass Fertilizer – A Pilot Project
Control
Algae
Commercial fertilizer
Measure:• Plant height• Circumference• Number of fruits/leaves
produced• Soil analyses
Other Algae Applications – Conclusions
• In addition to biofuel oils, algae produce many “high-value” products
• Some of the “high-value” products are billion dollar industries
• UT has developed technologies that can cost-effectively recover biofuel oils and high-value products
• Using CEM/OpenAlgae-developed technologies, we can collaborate with other research universities and private institutions to advance health applications
• The processed algae biomass is useful too -- we have initiated an algae fertilizer pilot program on the UT campus
• We continue to develop technologies useful to UT and industry
Contact Information
Dr. Rhykka ConnellyCenter for
ElectromechanicsResearch Scientist(512) [email protected]
Mr. Hoyt ThomasOpenAlgaePresident and CEO(713) [email protected]
Dr. Robert HebnerCenter for
ElectromechanicsDirector(512) [email protected]
