Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 33

lipid monitoring. Recently, a single-cell, laser-trapping Raman spectroscopic method
that is direct and in vivo has been described as an efficient tool for profiling microbial
cellular lipids (Wu et al., 2011). This method is proven in the quantitative estimation of
the degree of unsaturation and transition temperatures of algal cellular lipids.
3.6 PRESERVATION
3.6.1 t ransFer t eChniques
The accomplishment of bioprospecting rests with the successful long-term mainte-
nance of the algal strains. The most common method used to preserve microalgal
cultures is perpetual maintenance under a controlled environment. Periodic serial
sub-culturing of the mother culture onto agar slants is done to maintain the strains
(Day, 1999; Warren et al., 2002; Richmond, 2004). This provides metabolically
active cultures that retain a vigorous, morphologically, physiologically, and geneti-
cally representative population. A crucial factor to consider is that different dura-
tions of sub-cultures may provide different stages of the life cycle. Proper labeling
and careful checking are required before starting a serial transfer. Manipulations
and transfer should be carried out under aseptic conditions. Rigorous microbiologi-
cal methods, following standard guidelines for aseptic techniques and maintenance
procedures, are crucial (Isaac and Jennings, 1995). The revival of preserved cultures
can be successfully accomplished with 1% to 10% (v/v) of the original culture, but
some dinoflagellates, Synechococcus and Prochlorococcus , may require a higher
inocula level of up to 25% (v/v). Another issue with agar cultures is that some benthic
diatom colonies may stick rather firmly to the agar surface or that some filamentous
cyanobacteria may even grow into the agar. These can be transferred by removal of
agar along with algal material. If older agar cultures are to be revived, the slant may
be over-layered with fresh liquid medium for several hours prior to transfer. Another
issue that needs bearing in mind is that not all species form appreciable colonies
on agar, specifically many flagellates and other planktonic species; likewise, few
edaphic and aquatic benthic microalgae grow well in liquid medium. Regardless
of that, slant cultures are preferred because of easy and minimal handling during
transfer and hence a lower risk of contamination. Mixing the algal liquid culture is
customary during transfer to fresh medium or plates. However, uncontrolled mix-
ing bears the risk of damage to delicate coccoid green algae, cyanobacteria, and
some fragile diatoms such as Thalassiosira and Rhizosolenia. But mixing may be
mandatory in some instances or, as in case of Polytoma, where resting cells settle
to the bottom, the cell transfer should effect from the bottom of the culture vessel
(Anderson, 2005). In contrast, certain colonial flagellate and coccoid green algae
( Eudorina, Pediastrum ) need agitation and aeration to obtain typical morphology.
3.6.2 M aintenanCe C onditions
The maintenance of metabolically active algae is essential because of the conserva-
tion of stock cultures, attainment of explicit morphological and physiological sta-
tus, or mass production. As described earlier, conservation of stock cultures is by