Results from WP2 of BLUES: Preserving biodiversity: challenges and opportunities with biobank establishment and long-term storage of marine microbial isolates and invertebrate tissues.

Marine microorganisms and invertebrates present fundamentally different preservation challenges rooted in cellular complexity and physical parameters. Marine bacteria achieve substantially higher preservation success rates than invertebrate cells, with ocean bacteria maintaining 98.3% viability after 8 years in glycerol broth at -80°C and freeze-drying preserving 100% of bacterial strains over two years. The primary challenges for microorganisms involve contamination and genetic drift during long-term maintenance and variable tolerance to cryoprotectants. Sea cucumber cells have already been investigated for survival after freezing in Dorisol and standard media. Marine invertebrate tissues and cells face obstacles as high sensitivity to cryoprotectant toxicity, chilling sensitivity, etc. Sea cucumber Parastichopus tremulus individuals were collected as bycatch from commercial crustacean trawl and pot fisheries. The sampling for sea cucumber body wall associated microbiota has been done on three different sea cucumber batches, coming from two different locations. Different body wall tissues have been preserved in parallel. The sea cucumber culturable microbiota isolates, identified to the species level by Sanger sequencing, have been stored at different conditions and their viability checked every year for a 3-year storage. Body wall tissue samples were stored in two different media, at -20°C and -80°C as well as in liquid nitrogen and release of viable cells was monitored every month over a 3-month period. About 5% of the marine microorganisms stored at -80°C have lost viability after frozen storage for 3 years in 20% glycerol, while 3% have lost viability after frozen storage at -20°C in 20% glycerol. Viable cells were released into the cell culture medium after thawing in all storage condition cases. Numerous challenges exist regarding the correct maintenance and identification of marine environmental isolates for in-house culture collection establishment.