Perpetual transfer leading to long-term culturing, usually under conditions very dif­ferent from its natural environment, leads to genetic variants among the popula­tion 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 long­term 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 chemi­cals 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 preser­vatives in use today are formalin, 1% Lugol solution, and 3% glutaraldehyde (Wetzel 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 concentra­tion 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 dam­age. 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 rest­ing 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 Gottingen (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 rea­sons for this failure of cryopreservation on large and complex algae are not satisfy­ing. 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.