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Soil microbiomes are dynamic systems that respond to biotic and abiotic environmental factors such as those presented at seasonal scales or due to long-term anthropogenic regime shifts. These can affect the composition and function of microbiomes. Investigation of microbiomes can uncover hidden microbial roles in health and disease and discover microbiome-based interventions. Collections of soil samples are kept by various institutions in either a refrigerated or occasionally frozen state, but conditions are not optimised to ensure the integrity of soil microbiome. In this manuscript, we describe cryopreservation with a controlled rate cooler and estimate the genomic content of an exemplar soil sample before and after cryopreservation. The first hypothesis was to test the genomic integrity of the microbiome. We also enriched the soil sample with a liquid medium to estimate the growth of bacteria and compared their growth before and after cryopreservation. Sequence-based rRNA metabarcoding was used to demonstrate that the controlled rate cooler maintains intact the DNA content of the microbiome. Two methods of cryopreservation were applied and compared with control aliquots of soil. An optimised cryopreservation of soil samples is essential for the development of microbiome research in order to retain stable, functionally intact microbiomes. Our results showed that metabarcoding of 16S and ITS rRNA were useful methods to estimate successful cryopreservation. The soil microbiome after enrichment with liquid medium exhibited a similar response of cryopreserved soil and this was estimated with the comparison of the ten most abundant bacterial taxa. These findings support a successful process of cryopreservation and are promising for future use of this technology. To the best of our knowledge, this study is the first report of cryopreservation of soil using a Stirling cycle cooling approach.

Cryopreservation of a soil microbiome using a Stirling 1 cycle approach - a genomic assessment.