Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 31

extraction, for example, is the most common method for the determination of lipid
content in algae, but it requires a significant quantity of biomass (Bligh and Dyer,
1959; Ahlgren and Merino, 1991). Fluorescent methods using lipid-soluble dyes have
also been described, and although these methods require much less biomass (as little
as a single cell), it has not yet been established if these methods are valid across
a wide range of algal strains (De la Jara et al., 2003; Elsey et al., 2007). Further
improvements in analytical methodology could be made through the development
of solid-state screening methods. Not only are rapid screening procedures necessary
for the biofuels field, but they also could prove extremely useful for the identifica-
tion of species, particularly in mixed field samples necessary for the future of algal
ecology. They could also reduce the number of redundant screens of algal species.
3.5 SCREENING AND SELECTION FOR LIPID PRODUCTION
Conventional methods of solvent extraction and gravimetric determination for lipid
quantification (Bligh and Dyer, 1959) are laborious and time consuming. Moreover,
approximately 10 to 15 mg wet weight of cells (Akoto et al., 2005) must be cultured
for any appreciable extraction and derivatization. However, in-situ lipid content mea-
surements would significantly reduce the quantity of sample as well the preparation
time required. Accordingly, there is greater interest in a rapid in-situ measurement
of the lipid content of algal cells (Cooksey et al., 1987). Nile Red (9-diethylamino-
5H-benzo[α]phenoxazine-5-one), a lipid-soluble fluorescent dye, has been com-
monly used to evaluate the lipid content of animal cells and microorganisms such
as yeasts and fungi (Genicot et al., 2005) and specifically extended to microalgae
(Cooksey et al., 1987; Elsey et al., 2007). Nile Red is relatively photostable and pro-
duces intense fluorescence in organic solvents and hydrophobic environments, which
makes them a better candidate for in-situ screening for lipids. Furthermore, neutral
and polar lipids can be clearly differentiated due to polarity changes in the medium
as evinced by a blue shift in the emission maximum of Nile Red (Greenspan and
Fowler, 1985; Laughton, 1986; Cooksey et al., 1987; Lee et al., 1998). The solvent
system used for Nile Red would determine the emission spectra of the dye (Elsey
et al., 2007). However, the thick cell walls of microalgae inhibit the permeation of
Nile Red, and this is variable among algal species, requiring the use of high levels of
solvents such as DMSO (20% to 30% v/v) and elevated temperatures (40°C) (Chen
et al., 2009). Then again, Chen et al. (2011) developed a two-step microwave-assisted
staining method for in vivo quantification of neutral lipids in green algae with thick,
rigid cell walls that prevents penetration of the Nile Red dye into the cell. This may
also be appropriate for other classes of algae that do not stain properly with Nile Red.
Hence, a Nile Red assay can be used as a tool for screening oleaginous algal strains
as well as quantitatively determining the neutral lipids in algal cells (Figure 3.2; see
color insert).
Recently, another class of lipophilic fluorescent dye BODIPY ® 505/515
(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) has been used to
potentially stain microalgal lipids. BODIPY staining lets the lipid droplets stain green
and the chloroplasts stain red in live algal cells (Cooper et al., 2010). BODIPY 505/515
is advantageous over Nile Red in emitting a narrower spectrum (Cooper et al., 2010;