Context
Biological Ressources Centers (BRCs) are essential infrastructures supporting the Life Sciences and Biotechnology sectors (Janssen et al. 2010). Their establishment and maintenance require distributing well characterized, stable and performant biological materials (i.e. living organisms, cells, genes or related information) to the users in Life and Health sciences, Biotechnologies, Food industries, etc. Therefore, the implementation of reliable preservation technologies of the biological resources is crucial in the management of the BRCs.
Objectives
The project PRESPHOTO aimed to improve the preservation of photosynthetic microalgae (diatoms and cyanobacteria) as well as their genetic information in the BCCM/ULC and BCCM/DCG collections, respectively.
The major objectives of the project were:
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to improve cryopreservation methods for diatoms and cyanobacteria (higher viability and larger number of taxa),
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to evaluate the impact of the preservation protocols on the genomic stability of selected microalgal strains,
- to create and validate a genomic DNA bank of microalgae
Methodology (cyanobacteria)
The traditional two-step cooling and the encapsulation-dehydration methods were evaluated as potential long-term preservation techniques. The effects of several factors (e.g. culture conditions, the type and concentration of cryoprotectants (CPAs), preservation temperatures …) on the viability of four representative cyanobacterial strains using both cryopreservation methods have been first investigated to determine which are the most important for their successful cryopreservation. In addition, a vital staining method, allowing the rapid evaluation of post-cryopreservation viability was assessed for cyanobacteria. The selected cryopreservation protocols were further tested on 26 cyanobacterial strains. Finally, an independent validation of the developed protocols was performed by Culture Collection of Algae and Protozoa (CCAP, UK) (subcontractor) to ensure that they are robust, transferable and reproducible for the conservation of diatom and cyanobacterial strains.
The impact of cryopreservation on genomic stability of cyanobacteria is being studied. Three cyanobacterial strains for which a genome sequence is available is currently being resequenced to investigate the genetic changes induced by cryopreservation techniques.
In addition to the preservation as living strains, we also have focused on the DNA storage improvement. We have compared different DNA extraction, storage as well as quantity and quality control methods. In addition, for filamentous cyanobacteria, different pre-treatments were tested to disorganize the polysaccharidic sheaths that hinder DNA extraction.
Results (cyanobacteria)
Using the two-step cooling method, MeOH 5% (v/v) appeared the most effective CPA to preserve the four cyanobacterial strains in liquid nitrogen. The growth of the cyanobacterial cultures directly in the cryovials results in a faster recovery after cryopreservation. We suggested that during the growth the cultures may produce extracellular polysaccharides and some other molecules such as trehalose and glucose or antifreeze proteins that contribute to increased freezing tolerances (Tashyreva & Elster, 2012). In addition, growing the cultures in the cryovial avoids a centrifugation step which can damage cells (Bui et al., 2013). The developed two-step cryopreservation protocol was further tested on 26 cyanobacterial strains and resulted in 95% and 92% of viability after one day and one month of cryopreservation in LN. Non-cryopreserved cultures exhibited 95% of viability. In addition, the independent validation performed by the CCAP showed that this protocol is applicable to a range of filamentous cyanobacterial taxa.
For the encapsulation-dehydration, PEG-6000 was retained as the most appropriate CPAs as it results in low proliferation of the associated bacteria and allows the full recovery of most cultures. The evaporative dehydration with silica gel was more homogeneous and thus allows more reproducible results than dehydration under a laminar flow. Finally, the use of hexametaphosphate 3% is effective to dissolve the alginate beads and thus to promote the growth of the cyanobacterial cultures out of the beads after thawing and revival. However, the developed encapsulation-dehydration was not successfully validated on the 26 cyanobacterial strains as it resulted in 35% and 37% average viability after cryopreservation for one day and one month, respectively. Non-cryopreserved cultures exhibited 35% of viability. Low viability results were also observed by the CCAP in the validation experiments.
For DNA extraction, the highest yields were observed with the kit Nucleospin Tissue (Macherey-Nagel). This extraction kit has a protocol dedicated to the extraction of DNA from cyanobacteria. We showed that the extra buffer (containing 50 mM Tris/Cl (pH 8), 50 mM EDTA, 1 % (v/v) Triton X-100 and 20 mg/mL lysozyme) used to pre-lyse the cells was responsible for the increased yields. The use of different mechanical pre-treatments to disorganize the polysaccharidic sheaths from filamentous cyanobacteria did not significantly increase the yields of extracted DNA.
Conclusions and recommendations
Our results demonstrated that cryopreservation by the traditional two-step cooling procedures was suitable for cyanobacterial strains having various morphologies and origins while the encapsulation-dehydration requires further major improvements before the adoption of this method for the maintenance of cyanobacterial strains, especially the encapsulation and the dehydration steps of the protocol.
The developed two-step cryopreservation protocol has been included in the ISO 9001 QMS and cyanobacterial strains of the collection are progressively preserved using this protocol. In addition, DNA from cyanobacteria will be extracted using the developed extraction method.
The improvement of the preservation techniques and the creation of a DNA bank in the two BCCM collections of microalgae will improve their capacity to fulfill the needs of their users. Indeed, photosynthetic microalgae, including diatoms and cyanobacteria, are increasingly used in different areas of applied research, as in biotechnology (e.g. biodiesel, bioplastics), pharmaceutical research (e.g. bioactive molecules), or cosmetic applications. They are also used as food complement (polyunsaturated fatty acids), bio-fertilizers, bio-pesticides, animal food, etc. Their use in these fields requires having access to high-quality and authentic biological resources.