| dc.description.abstract | The establishment of culture collections is a vital aspect for the conservation
freshwater microalgae, which are a very diverse group of organisms with important
ecological functions on the ecosystems maintenance and an immense although still sparsely
explored biotechnological potential, particularly for the use of metabolites and biodiesel
production. The traditional protocol for culture maintenance is the serial subculturing of
metabollicaly active cultures. Although strains have been maintained in metabolically active
cultures for decades, there are several reports of loss of characteristics for the organisms
thus maintained, and an increase in the number of strains could be the cause for logistical
challenges on the maintenance regimes and elevated costs with material and functionalism.
Cryopreservation, which is the maintenance of organisms at ultra-low temperatures to the
point that all cellular metabolic functions are paused, appears as a recommended alternative
for the maintenance of long-term culture collections. The use of this technique allows the
maintenance of samples for long periods of time with minimal handling, severely reducing
the maintenance costs, while maintaining the stability of cultures, which should be stored at
ultra-low temperatures to eliminate the occurrence of intracellular chemical processes.
The Freshwater Microalgae Culture Collection (CCMA-UFSCar) currently maintains
approximately 700 microalgae strains in metabolically active cultures, which is reaching
maximum support capacity for routine maintenance practices. The requirements with
cultures maintenance will also, consequently, affect the provision services of the collection,
which are the basis of research of a large number of projects throughout Brazil. Thus, the
main objective of this project was to test the feasibility of the use of cryopreservation as a
technique to maintain algal cultures in this culture collection. For this, initially positive
results were obtained for the application of a standard freezing protocol, particularly for
green coccoid algae. However, larger and more complex organisms are still recalcitrant to
freezing, with low rates of recovery, which will require further research and adaptation of
the protocols to meet the particularities of each strain. In parallel to the establishment of
the cryopreserved biobank for CCMA-UFSCar, projects were developed in order to further
clarify details and consequences of the process of freezing and storing organisms at low
temperatures, seeking to improve the success rates for the implementation of this
technique.
The effects of the presence of contaminating organisms on the microalgae cultures
on the response of strains to freezing, and consequently on the formation of viable and
robust post-thaw cultures, were verified a few selected non-axenic strains. It was noticed
that the choice of cryoprotectant solution (CPA), used to protect cells during freezing, may
be crucial for post-thaw recovery, and it is necessary to search for balance between
obtaining the highest possible viability levels and avoiding the extreme proliferation of
contaminating organisms, which could lead to inhibition in the recovery and quality of the
desired algal cultures after the process.
In addition to direct survival to the freezing process, which is a potentially damaging
for cells, the longevity of samples maintained at different temperatures was also tested,
aiming to establish the simplest and most cost-effective procedure to ensure the long term
survival of frozen samples. It was observed that the storage temperatures, as well as the CPA
used during freezing, were significant aspects to guarantee the continued viability of the
frozen cultures, and only samples maintained immersed in liquid nitrogen were sufficiently
protected against temperature fluctuations, ensuring the survival of all the samples for
longer periods. Furthermore, the continuity of the viability for samples maintained
immersed in liquid nitrogen was also observed in a study with samples maintained on these
conditions for up to 40 years at Culture Collection of Algae and Protozoa (CCAP, UK).
The freezing process per se is potentially damaging to the cultures during the process,
which might be the cause for functional and genetic differences in cells that have undergone
this process. Thus, biochemical and genetic analysis were used to verify the stability of the
cultures submitted to the freezing protocol.
To ensure the successful implementation of a cryopreserved microalgae bank for
CCMA-UFSCar, in addition to viability and stability measures, it is necessary to establish
protocols for sample management and routine maintenance. Thus, a case study was carried
out with the experiences acquired in CCAP during the 40 years of the history of a
cryopreserved bank, and how the application of these techniques can benefit the quality
control and management of CCMA-UFSCar, facilitating the implementation of the
cryopreserved biobank for this culture collection. | |
