Cryopreservation and recovery of a complex hypersaline microbial mat community

Cryopreservation of microorganisms is an essential tool in industrial- and food applications where conservation of microbial activity and critical beneficial traits need to be guaranteed to provide a consistent product or production process. This often refers to simple, single species or low diversity assemblages in liquid cultures that can easily be revived and regrown to perform the desired process. Cryopreservation is also of essence for scientific experimentation where many environmental samples are taken in remote sampling sites and at high costs. Biobanking, or the long term preservation and potential revival of complex, structured samples come with an additional challenge related to maintaining the structure upon revival. Here we look at cryopreserving and reviving a complex photosynthesis driven microbial mat from a hypersaline ecosystem. Amplicon sequencing of the 16S and 18S ribosomal RNA gene was used to determine the community composition of bacteria and eukaryotes respectively. The tests included the use of different cryopreservative agents and different times of cryopreservation at -150 °C. Upon revival, the cryopreservatives cannot be separated from the preserved samples without disturbing the community structure, while carryover of these compounds may influence reconstitution of the communities. Indeed, although both glycerol and Me₂SO are good cryopreservatives of microbial assemblages, carryover of these compounds had a profound negative effect on the reestablishment of a functional microbial mat. Best cryopreservation and reconstitution results were obtained in the absence of a cryopreservative agent or when methanol was used.