Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 27

separate the larger and heavier cells from smaller algae and bacteria. Specifically
for large dinoflagellates and diatoms, moderate centrifugation for a short duration is
enough to pelletize them, and smaller cells can be decanted. Density gradient cen-
trifugation with silica sol, Percoll™, etc. has been successfully employed to separate
mixed laboratory cultures so that individual species can be separated into a sharp
band (Reardon et al., 1979). Large, nonmotile algal cells can be effectively separated
by settling. Hence, gravimetric settling is the choice if one aims for concentrating
larger cells; however, it is not effective to obtain unialgal culture and hence suggests
some combination with other procedures.
3.3.3 a dvanCed M ethods
Although single-celled, colonial, or filamentous algae growing on the agar surface
can be isolated by streak plate or spraying, any flagellates as well as other types of
algae require the use of advanced techniques. A unialgal culture would contain only
one kind of alga, usually a clonal population, but may contain other life forms such
as bacteria, fungi, or protozoa. Alternatively, cultures may be axenic, in that they
contain only one species of alga. Unialgal cultures are best isolated by targeting the
isolation of the zoospores immediately after release from parent cell walls as those
cells that begin attaching to surfaces are likely to add contaminants. The algal isola-
tion techniques involving cell separation pose limitations with highly heterogeneous
samples or when the cells are suspended in a solution of different chemicals, biomol-
ecules, and cells. This can be overcome by employing a micromanipulator, which
successfully permits the separation of a single cell from a liquid culture. The single
cell can be easily separated from an enriched environmental sample and grown in
liquid medium as monoculture or in agar plates, thus facilitating a significant time
saving over the conventional plating technique. The micromanipulator is the ideal
tool for algal screening and isolation, provided the person handling it has acquired
skill in handling the equipment (Kacka and Donmez, 2008; Moreno-Garrido, 2008).
Using micromanipulation techniques requires expertise and skill. It requires the
handling of an inverted microscope or stereo zoom microscope with a magnification
up to 200×. Phase contrast or dark-field microscopy offers advantages. Capillary
tubes or hematocrit tubes of approximately 1 mm diameter × 100 mm long are used
for picking individual cells (Godhe et al., 2002; Knuckey et al., 2002).
High-throughput cell sorting is possible when coupled with flow cytometry, which
facilitates the rapid and efficient screening of microalgal strains. Microalgae possess
different photosynthetic pigments, emitting various auto-fluorescence, which can be
applied in flow cytometry to identify algae (Davey and Kell, 1996). Literature on
the isolation of microalgae from natural waters employing flow cytometric cell sort-
ing is available (Reckermann, 2000; Crosbie et al., 2003). Chlorophyll is used as a
fluorescent probe to distinguish different strains of microalgae. Reckermann (2000)
and Sensen et al. (1993) used the chlorophyll auto-fluorescence (CAF) properties of
eukaryotic phytoplankton, diatoms, and pico-autotrophic cells for isolation of axenic
cultures, whereas Crosbie et al. (2003) used both red and orange auto-fluorescence
to differentiate species of algae. Similarly, green auto-fluorescence (GAF), which
is common in both autotrophic and heterotrophic dinoflagellates, is also a valuable