Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids from live cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GC-FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GC-MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived from the residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae; the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris. The method is shown to be robust, highly reproducible, and fast, allowing for multiple samples to be analyzed throughout the time course of culturing, thus providing time-resolved information regarding lipid quantity and quality. Total time from harvesting to obtaining analytical results is less than 2. h.
Bibliographical noteFunding Information:
This material is based upon work supported by the National Science Foundation (NSF) under CHE-1230632 . A portion of this research was supported by the U.S. Department of Energy (DOE) Office of Energy Efficiency and Renewable Energy (EERE) Biomass Program under Contract Nos. DE-EE0003136 and DE-EE0005993 . Support for EL and RDG was also provided by the NSF IGERT Program in Geobiological Systems ( DGE 0654336 ). Instrumental support was provided through the Center for Biofilm Engineering (CBE) at Montana State University (MSU) as well as by the Environmental and Biofilm Mass Spectrometry Facility (EBMSF) funded through DURIP Contract Number: W911NF0510255 and the MSU Thermal Biology Institute from the NASA Exobiology Program Project NAG5-8807 .
- Fatty acid methyl ester (FAME)
- Nile Red fluorescence
- Triacylglycerol (TAG)