Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 36

and Likens, 2000). The concentration can be altered based on the type and nature of
the algae and maintenance conditions. For instance, a 3% glutaraldehyde concentra-
tion is too high, causing withering or complete disintegration of cells beyond the
ability for retention of normal cell shape, specifically of wall-less flagellates.
To overcome the shortcomings and inclusion of chemicals in maintenance
medium, lyophilization has been accepted widely as a means of conserving viable
cultures of all microorganisms in a desiccated state. However, lyophilization involves
vacuum desiccation under freezing and subsequent thawing, so cell revival mandates
inclusion of cryoprotective agents at high concentrations to offer protection from dam-
age. The cryoprotectants extensively used for algae are methanol, dimethylsulfoxide
(DMSO), and glycerol (Taylor and Fletcher, 1998). Methanol and DMSO are preferred
for freshwater and terrestrial microalgal cryopreservation, while glycerol and DMSO
are useful for marine phytoplanktons (Day et al., 2000). The above are penetrating cryo-
protectants and passively move through the plasma membrane to equilibrate between
the cell interior and the extracellular solution. Penetrating cryoprotectants are toxic at
high concentrations (Adam et al., 1995; Santarius, 1996). Hence, permeating cryopro-
tectants should be added prior to cryopreservation and should immediately be removed
after thawing. Algal spore preservation is heavily dependent on bacterial contamination.
Hence, preservation of spores of the green seaweeds Ulva fasciata and U. pertusa was
improved by the addition of ampicillin in f/2 medium at 4°C (Bhattarai et al., 2007).
Cryopreservation is most suited for algae that do not require that the normal rest-
ing stage be maintained indefinitely. Because microalgae are cryopreserved as large
populations of algal units, the percentage of viability of identical cultures is of great
concern and often varies. However, with proper physiological conditioning prior to
freezing, the variability can be minimized. This is one of the key reasons that, to
date, most dinoflagellates, cryptophytes, synurophytes, and raphidophytes are not
successfully cryopreserved. In contrast, most marine diatoms can be effectively
cryopreserved, with high viability, although freshwater diatoms fail to revive and
have thus proven more problematic. Examinations of large numbers of strains have
taken place at the four major protistan collections: Culture Collection of Algae and
Protozoa (CCAP) (United Kingdom), The Provasoli-Guillard National Center for
Culture of Marine Phytoplankton (CCMP) (United States), Sammlung von Algenku
Huren Göttingen (SAG) (Germany), and The Culture Collection of Algae at UTEX
(United States); examination reveals that chlorarachniophytes, eustigmatophytes,
pelagophytes, phaeothamniophytes, and ulvophytes also have very high success
rates, comparable with the other green algae and cyanobacteria. Algal strains that
have been reestablished at NREL are being cryopreserved in an effort to reduce the
workload associated with maintaining an algae collection and to prevent unintended
loss or genetic drift, a risk associated with frequent transfer. The cryofreezer uses
liquid nitrogen, and cultures are stored at −195°C in the vapor phase. Nevertheless, it
has been distinguished that virtually all large cell sized algae, and most filamentous
forms, cannot as yet be cryopreserved. Attempts to determine the fundamental rea-
sons for this failure of cryopreservation on large and complex algae are not satisfy-
ing. This warrants auxiliary research on the basic mechanisms of freezing damage.
Furthermore, the pragmatic development of improved techniques will expand the
number and diversity of algal taxa that can be successfully cryopreserved.