Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples

<div><p>Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.</p> </div

Principal coordinates plots of all Earth Microbiome Project sequenced soil samples based on unweighted UniFrac (A), weighted UniFrac (B), and Bray-Curtis (C) distances.

<p>The samples analyzed for this study are represented by open red triangles. All other samples are colored by biome: blue = polar desert, green = tundra, orange = temperate, black = warm desert, cyan = tropic. Filled triangles = EMP study 632, filled squares = 659, inverted open triangles = 722, inverted filled triangles = 1035, filled circles = 808, open diamonds = 1031, open squares = 1034, open circles = 1036, '+’s = 1037, 'X’s = 1038, and'*’s = 1526.</p

Mean ± standard error of: (A) and (B) evenness (equitability) between storage times and storage temperatures, respectively; (C) and (D) unweighted UniFrac distances between samples within each time and temperature treatment, respectively.

<p>Mean ± standard error of: (A) and (B) evenness (equitability) between storage times and storage temperatures, respectively; (C) and (D) unweighted UniFrac distances between samples within each time and temperature treatment, respectively.</p

Ternary plot of distribution of bacterial taxonomic groups among sample types in the second experiment.

<p>Size of circles proportional to the log of the total abundance, taxonomic groups are all phylum-level, except for Proteobacteria, which is by class.</p

PCoA plots of microbial community similarity in first experiment for unweighted analysis (A–B) and weighted analysis (C–D).

<p>Plots for unweighted analysis are based on unweighted UniFrac distance, and demonstrate relationship between sample type (A), strain (B), and the major PC axes (PC 1 = 26.46% variance, PC 2 = 7.36% variance). Plots for weighted analysis are based on weighted UniFrac distances, and demonstrate relationship between sample type (C), strain (D), and the major PC axes (PC 1 = 62.98% variance, PC 2 = 15.43% variance). Abbreviations for strains are denoted by B (Burmese), BK (BooKoo Kush), and D (Sour Diesel).</p

Box plots of alpha diversity (observed species) for endorhiza, rhizosphere, and bulk soil from two separate soil types in the second eperiment.

<p>MB  =  Mo-Bio soil, OC  =  Orange County soil. Note the significant differences between alpha diversity in the bulk soil and rhizosphere but negligible differences between endorhiza alpha diversity between soil types.</p

Soil Physicochemical Data.

<p>Physical composition and tested edaphic factors for five soil types from both experiments. Abbreviations for Soil ID are: MB indicates Mo-Bio soil, OC indicates Orange County soil, number indicates experiment (1 =  first experiment, 2 =  second experiment), and final letter abbreviations detail the associated cultivar with the bulk soil. B =  Burmese, SD =  Sour Diesel, BK =  Bookoo Kush.</p

Box plots of beta-diversity distances between communities for both weighted and unweighted analyses.

<p>Initials (i.e. B vs. C) stand for comparisons of beta-distances for samples within groups (R =  rhizosphere, C =  <i>Cannabis</i> endorhiza, B =  bulk soil).</p

PCoA plots of microbial community similarity in second experiment for unweighted analysis (A–C) and weighted analysis (D–F).

<p>Plots for unweighted analysis are based on unweighted UniFrac distances, and demonstrate relationship between soil type (A), sample type (B), strain (C), and the major PC axes (PC 1 = 32.06% variance, PC 2 = 11.34% variance, PC 3 = 5.67% variance). Plots for weighted analysis are based on weighted UniFrac distances, and demonstrate relationship between soil type (D), sample type (E), strain (F), and the major PC axes (PC 1 = 34.51% variance, PC 2 = 25.41% variance, PC 3 = 19.31% variance). Note that PC 1 in the unweighted analysis is dominated by variation in soil type (A), but PC 1 in weighted analysis is dominated by strain (F). Grey points (Fig. 2c, 2f) represent bulk soil samples that aren't associated with either strain. Abbreviations for strains are denoted by MW (Mauie Wowie) and WW (White Widow), and abbreviations for soil type are denoted by MB (Mo-Bio soil) and OC (Orange County soil).</p