Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 35

a key factor. For routine maintenance, sub-culturing is done toward the end of the
exponential growth phase of the culture. The shortest transfer cycle is about 1 to
2 weeks for sensitive strains, while some green algae and cyanobacteria, on agar
slants at low light and 10°C, is sub-cultured only once every 6 months. However,
a safe transfer interval for a specific strain can be predictable to one quarter the
time a strain can survive maximally. Usually, a post-transfer period at higher light
and temperature regime is valuable in regular quality control assessment. Moreover,
perpetual maintenance over longer periods may alter the morphological features and
physiological characteristics of some strains, and hence a short interval of mainte-
nance at optimal growth conditions is recommended to refresh the culture. In case
of an unknown or a newly isolated strain, the cultural characteristics should be fully
understood prior to maintenance, or the long-term maintenance procedure should
be framed by optimization of survival at varied light and temperature conditions
(Lokhorst, 2003). It is often sensible to screen and configure a suitable medium for
the new isolate. Finally, the culture maintenance chamber or room should be con-
trolled for humidity—for not only preventing evaporation of cultures, but also to
avoid the contaminating fungi and molds.
In spite of these, the selective and synthetic nature of the media as well the incu-
bation conditions, as opposed to the native ecological conditions, limit the success of
perpetual transfer. Furthermore, continuous transfers lead to the loss of morphologi-
cal and/or genetic characteristics (Warren et al., 2002). Not to mention that serial
sub-culturing is a labor-intensive and time-consuming process, which restricts main-
tenance and handling of a large number of cultures. Above all, to overcome the risk
of loss of strains, each strain may be maintained in secondary culture collections
with sub-cultures of different ages or transfer dates. The World Federation of Culture
Collections has suggested to stock backup cultures at various locations to expedite
all possible chances of revival (Anon, 1999).
3.6.3 C ryopreservation
Perpetual transfer leading to long-term culturing, usually under conditions very dif-
ferent from its natural environment, leads to genetic variants among the popula-
tion adapted to the artificial culturing environment. To sidestep the shortcomings
of serial sub-culturing, alternate methods of ex-situ conservation of algal strains
are suggested. Continuous maintenance of actively growing algal strains on a long-
term basis is often costly, and time and labor consuming. In contrast, cultures can
be maintained alive in a retarded metabolic state that requires less attention. One
approach is to maintain resting spores or other dormant stages of some algal species
(such as akinetes) at ambient temperatures for many years without any attention.
Leeson et al. (1984) were able to recover aplanospores of Haematococcus pluvialis
Flotow from air-dried soil even after 27 years. However, it should be considered that
the viability of resting stages generally declines with time, and many aquatic algae
do not show any insistent dormant stage. Hence, the addition of bacteriostatic chemi-
cals and agents that prevent autolysis of algal cells to help improve the cell viability
during the entire storage time is generally recommended. Some of the major preser-
vatives in use today are formalin, 1% Lugol solution, and 3% glutaraldehyde (Wetzel