Triacylglycerol profiling of marine microalgae by mass spectrometry

Abstract

ingly important. The methods and pursuit of algae-based fuels also have applications for bioactive lipids and highvalue, lipid-based chemical precursors ( 4,5 ), including polyunsaturated fatty acids (PUFA), carotenes, antioxidants, vitamins, and pigments, which have applications in the pharmaceutical, nutraceutical, and food industries. In all such applications, the methods of analysis, access to desired composition, total yield, and effi ciency with which lipids can be converted to usable products will be the ultimate determinants of industrial success.Previous analysis of algae lipid composition using GC/MS and MALDI-TOF mass spectrometry (MS) has shown a broad variety of lipid classes: glycerolipids, such as monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG); phospholipids, such as phosphatidylcholine (PC); and neutral lipids (triacylglycerols) ( 6-9 ). Many reports have focused on the analysis of polar lipids, but TAG analysis and composition have become increasingly important for biofuel applications. TAG analysis of mixed samples remains a challenge because of its lower ionization efficiency relative to polar lipids (such as phospholipids) ( 10 ). Analysis of neutral lipids in microalgae often relies on lipophilic fl uorescence and colorimetric assays ( 11, 12 ); however, these assays do not provide compositional TAG characterization. Compositional analysis of microalgal TAG primarily involves the initial separation of extracts using either liquid chromatography or thin-layer chromatography followed by MS analysis or derivatization of extracts, such as transesterifi cation, followed by analysis of fatty acid methyl esters (FAME) by GC/MS ( 13-15 ). The direct analysis of algal triacylglycerol using pyrolysis GC/MS has been reported as a promising alternative, but the purchase of specialty equipment and use of higher temperature ranges may limit the broad utility of this method ( 16,17 ).Herein, we describe the compositional analysis of intact triacylglycerol of oleaginous marine algae using MALDI-TOF MS. MALDI-TOF provides a direct method Abstract We present a method for the determination of triacylglycerol (TAG) profi les of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species ( Phaeodactylum tricornutum , Nannochloropsis salina , Nannochloropsis oculata , and Microalgae have been identifi ed as an attractive source for the production of biofuels in addition to bioactive lipids and high-value, lipid-based chemical precursors based on their ability to produce 20% or more of their mass as oil. Due to the conversion of triacylglycerols (TAG) to fatty acid alkyl esters for use as biodiesel ( 1, 2 ), current research has focused on TAG production and analysis to make biofuels from microalgae a more economically feasible option ( 3 )....

Exploring Valuable Lipids in Diatoms

“…Generally, the cartridge needs to be conditioned with hexane, and neutral lipids, glycolipids, and phospholipids will be eluted stepwise using chloroform, acetone, and methanol according to the polarity of each fraction. The use of hexane instead of chloroform for the conditioning and elution of neutral lipids has also been reported in order to avoid toxicity of chloroform (Danielewicz et al, 2011). It should be noted that phosphatidylcholines, the cholinecontaining phospholipids, might be retained in the SPE column, which could be verified by comparative profiling of each fraction with raw lipid extracts (Danielewicz et al, 2011).…”

“…Hexane extraction results in decreased yields of polar lipids due to its non-polar property. However, the use of hexane would reduce the amount of chlorophyll, which is a major contaminant for lipid extraction (Danielewicz et al, 2011). Different aforementioned solvents have been studied for lipid extraction, and it has been found that chloroform-methanol (1:1) is optimal for a total lipid recovery (Ryckebosch et al, 2012).…”

“…In order to reduce the solvent toxicity from chloroform, alternative solvents or solvent pairs have also been tested, such as methyltert-butyl ether (Matyash et al, 2008), hexane (Danielewicz et al, 2011), and hexane/isopropanol (Hara and Radin, 1978). Hexane extraction results in decreased yields of polar lipids due to its non-polar property.…”

“…On the advent of solid-phase extraction (SPE) cartridges, lipid classes in diatoms were fractionated using a silica-based SPE method (Yongmanitchai and Ward, 1992). This method is followed by many later studies (Chen et al, 2007;Danielewicz et al, 2011;Ryckebosch et al, 2012). Generally, the cartridge needs to be conditioned with hexane, and neutral lipids, glycolipids, and phospholipids will be eluted stepwise using chloroform, acetone, and methanol according to the polarity of each fraction.…”

High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product

“…8,9) In recent years, various strains of Nannochloropsis have been investigated for their biomass production, lipid composition, and content under different growth conditions. 8,[10][11][12][13][14] To further develop them into an integrated photons-to-fuel production platform, high-efficiency transformation methods are necessary.…”