Preservation and storage effects on river sediment microbiomes: Implications for community stability and ecological inference

Sample preservation and storage can alter microbial communities between sampling and analysis, influencing the interpretation of environmental microbiome data. We investigated how preservation method, storage temperature, and storage duration affect river sediment microbiomes using 16S and 18S rRNA gene amplicon sequencing. Sediments were preserved in ethanol, nucleic acid preservation (NAP) buffer, or without a preservative, and stored at room temperature or frozen at -20 degrees Celsius or -80 degrees Celsius for up to eight weeks. We report that preservation method, storage conditions, and their interaction influenced estimates of microbial diversity and community composition. Differences between treatments were detectable after approximately 18 h and generally increased with storage time. Frozen samples remained closest to their treatment-specific baseline communities, whereas room-temperature storage, particularly without a preservative, produced the largest changes. Notably, ethanol and NAP buffer preservation reduced, but did not prevent, changes during room-temperature storage. Preservation also influenced taxonomic patterns and the outcome of phylogenetic null-model analyses. Frozen storage generally retained the sample's assembly-metric estimates, with stronger signatures of deterministic community assembly, whereas room-temperature storage shifted the inferred balance toward stochastic processes. Our results show that preservation and storage conditions can affect both the microbial community measured and the ecological conclusions drawn from it. Among the conditions tested, frozen storage provided the best preservation of sediment microbial communities and should be preferred when samples cannot be processed immediately.