fraction of algae as the total quantity of compounds soluble in a
chloroform/methanol solvent mixture but also has led to challenges
in the interpretation of these data [3–5]. A multitude of reports are
present in the literature assessing the application of solvent extraction
processes, where it has been shown that gravimetric extraction yield
for lipids is highly dependent on the polarity of the solvents used
and the composition of the algal lipids [6–8]. The utilization of an
extraction-based quantification of the lipid fraction is thus fraught
with challenges, and the respective yields obtained are highly dependent on the species and nutritional status of the cells [9].
In situ or direct transesterification of whole microalgal biomass is
an alternative to lipid quantification and is routinely used to determine total lipid content in whole biomass samples. This method
eliminates the need for timely and noncompound-specific extraction
gravimetric-based analyses. Several methods exist for direct transesterification of biomass and lipid samples [10–14]. These methods
typically rely on a base, acid, or base/acid two-step catalysis to transesterify lipids. Base catalysts exhibit lower yields compared to acid
catalysts in the presence of free fatty acids [13]. Due to the potential
presence of free fatty acids in microalgal samples, we chose to focus on
the use of an acid catalyst, which is known to be more effective at
conversion of all lipid types [10, 13]. Methanolic HCl is a commonly
employed catalyst for esterifying lipids in situ, and its effectiveness on
microalgal lipids has been studied on both large and small scales. We
have shown that it is an effective catalyst for the small-scale esterification of typical microalgal lipids within a biomass matrix, even in the
presence of high percentages of water [13]. Using chloroform/
methanol as a cosolvent and HCl/MeOH as a catalyst, we can
transesterify microalgal lipids into their fatty acid methyl ester
(FAME) equivalents on a scale requiring less than 2 mL of total
reagent/solvent and 10 mg of biomass. We describe here the process
of transesterification, with a detailed description of quantification of
the respective FAME by gas chromatography (GC).
2 Materials
Prepare all reagents in a fume hood. Use solvent-compatible analytical ware when completing all steps. Store reagents in a suitable
location for flammable and toxic chemicals. Carefully follow all
relevant chemical handling procedures.
2.1 Apparatus
1. Analytical balance, accurate to 1 mg or 0.1 mg.
2. Vacuum oven set to 40 Æ 3
C or a vacuum desiccator.
3. Digital dry block, capable of maintaining 85 Æ 3
C.
4. Volumetric flask (class A), 10 mL.
204
S. Van Wychen and L.M.L Laurens
chloroform/methanol solvent mixture but also has led to challenges
in the interpretation of these data [3–5]. A multitude of reports are
present in the literature assessing the application of solvent extraction
processes, where it has been shown that gravimetric extraction yield
for lipids is highly dependent on the polarity of the solvents used
and the composition of the algal lipids [6–8]. The utilization of an
extraction-based quantification of the lipid fraction is thus fraught
with challenges, and the respective yields obtained are highly dependent on the species and nutritional status of the cells [9].
In situ or direct transesterification of whole microalgal biomass is
an alternative to lipid quantification and is routinely used to determine total lipid content in whole biomass samples. This method
eliminates the need for timely and noncompound-specific extraction
gravimetric-based analyses. Several methods exist for direct transesterification of biomass and lipid samples [10–14]. These methods
typically rely on a base, acid, or base/acid two-step catalysis to transesterify lipids. Base catalysts exhibit lower yields compared to acid
catalysts in the presence of free fatty acids [13]. Due to the potential
presence of free fatty acids in microalgal samples, we chose to focus on
the use of an acid catalyst, which is known to be more effective at
conversion of all lipid types [10, 13]. Methanolic HCl is a commonly
employed catalyst for esterifying lipids in situ, and its effectiveness on
microalgal lipids has been studied on both large and small scales. We
have shown that it is an effective catalyst for the small-scale esterification of typical microalgal lipids within a biomass matrix, even in the
presence of high percentages of water [13]. Using chloroform/
methanol as a cosolvent and HCl/MeOH as a catalyst, we can
transesterify microalgal lipids into their fatty acid methyl ester
(FAME) equivalents on a scale requiring less than 2 mL of total
reagent/solvent and 10 mg of biomass. We describe here the process
of transesterification, with a detailed description of quantification of
the respective FAME by gas chromatography (GC).
2 Materials
Prepare all reagents in a fume hood. Use solvent-compatible analytical ware when completing all steps. Store reagents in a suitable
location for flammable and toxic chemicals. Carefully follow all
relevant chemical handling procedures.
2.1 Apparatus
1. Analytical balance, accurate to 1 mg or 0.1 mg.
2. Vacuum oven set to 40 Æ 3
C or a vacuum desiccator.
3. Digital dry block, capable of maintaining 85 Æ 3
C.
4. Volumetric flask (class A), 10 mL.
204
S. Van Wychen and L.M.L Laurens