Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 23

several times with sterile saline to remove bacteria and then inoculate to BG-11
medium (Rippka et al., 1979) for enrichment. However, some algal strains take
weeks to months to be isolated by traditional methods (Anderson, 2005). For
large-scale sampling and isolation efforts, high-throughput automated isolation
techniques involving fluorescence-activated cell sorting (FACS) have proven
extremely useful (Sieracki et al., 2005). Because of morphological similarities
when comparing many algal species, actual strain identification should be based
on molecular methods such as rRNA sequence comparison or, in the case of
closely related strains, other gene markers.
3.3.1 M edia C onFiGuration
The distribution of algal species is facilitated by both the selective action of the
chemo-physical environment and by the organism's ability to colonize a particular
habitat. Numerous culture media have been developed for the isolation and cultiva-
tion of microalgae. Some of them are modifications formulated based on the nutri-
ent requirements of the organism. For instance, better growth of marine algae can
be achieved by adding small quantities of natural seawater (less than 1% to 4%)
rather than supplementing with artificial seawater. Likewise, Schreiber solution,
a mixture of nitrate and phosphate, was based on the minimum requirement of the
two elements by a diatom culture. Soil extract is amended to Schreiber's medium for
cultivating green dasycladalean Acetabularia and some unicellular benthic marine
algae. Earlier algal media were formulated, to include antibiotics, vitamins, trace
metals, and organic chelators such as citrate, which was later replaced with EDTA.
Likewise, Chu's medium No. 10 was composed based on proximate analyses of natu-
ral samples such as eutrophic lakes. Antibiotics are generally added to the growth
medium to inhibit contaminating protists. Germanium dioxide is suggested to inhibit
the growth of diatoms. Antibiotics are also helpful as extensive cleansing agents.
McDaniel et al. (1962) were able to purify algal cultures free of bacterial contami-
nation using a procedure involving treatment with a detergent and carbolic acid.
Various media configured for isolation and cultivation of algae are given in Table 3.4.
3.3.2 t raditional M ethods
Using a micropipette or Pasteur pipette, or a glass capillary having a straight, bent,
or curved tip is handy for single-cell isolation. Micropipettes enable fishing out a
single cell from the sample after a series of transfers into sterile rinsing droplets,
without the cell being damaged in the process. Finally, the single cell can be pipetted
and transferred to the culture medium after microscopic examination. Lewin (1959)
recommended placing the droplets on agar to reduce evaporation, but this depends
on the size of the cells. Technical skill and expertise are important in order not to
shear or damage the cell. The damage may be apparent as cessation of swimming
in flagellates or a difference in light refraction due to broken frustules as in diatoms,
and severe damage is evident by leakage of protoplasm. The traditional method of
micropipette isolation can be successfully attempted with the use of ultra-pure sterile
droplets for rinsing, as marine samples hold suspended particles.