16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

<div><p>A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities.</p></div

Alpha diversity of Winogradsky column samples.

<p>Alpha diversity indices were calculated on rarefied samples. Samples were pooled by layer (A,B,C) or by sediment source (D,E,F) and the average Shannon index (A,D), richness (B,F) and Berger-Parker dominance index (C,F) were calculated. Error bars represent standard error for each category. Significant differences were seen between the Shannon index of top surface samples and samples taken from 4, 8, and 12 cm below the surface (A, non-parametric t-test, p = 0.021) and between sediment sources (D, nonparametric t-test, p = 0.001). Top surface samples were significantly less rich than samples at 4, 8, and 12 cm below the SWI (B, non-parametric t-test, p = 0.021 for surface vs. 4 cm, p cm, p = 0.021 surface vs. 8 cm, and p cm, and p = 0.042 for surface vs. 12 cm cm) and Eph's Pond columns are significantly more rich than Buxton Pond columns (E, non-parametric t-test, p = 0.002). Buxton Pond columns were significantly more dominated by single taxa than Eph's Pond columns (F, non-parametric t-test, p = 0.015).</p

Cladograms of biomarkers for depth, sediment source, and cellulose source.

<p>LEfSe was used to identify biomarkers that discriminate (A) depth, (B) sediment source, and (C) cellulose source in Winogradsky columns. Concentric rings from outside in are genus, family, order, class and phylum, with the two central circles for bacteria and archaea. All taxa present are shown, with colored circles representing biomarkers and yellow circles representing non-discriminating taxa. Shaded areas show all taxa below phylum or class biomarkers. For clarity, only biomarkers at the phylum and class levels are labeled.</p

Sulfur cycling organisms identified in Buxton Pond and Eph's Pond Winogradsky column communities.

<p>Heat maps show the relative abundance of abundant sulfur and sulfate reducers (left) and sulfur or sulfide oxidizers (right). Samples are ordered by depth from top to bottom. *Eph's Pond column biomarkers, †Buxton Pond column biomarkers. GSB: green sulfur bacteria, PNSB: purple non-sulfur bacteria, CL: chemolithotroph, PSB: purple sulfur bacteria.</p

UPGMA trees of all Winogradsky column samples show separation by depth and sediment source.

<p>Rarefied weighted UNIFRAC results were used to generate a consensus UPGMA tree. Samples are colored by (A) sediment source, (B) depth, and (C) column.</p

Principal coordinate analysis of Winogradsky column beta diversity.

<p>Principal Coordinate plots of weighted UNIFRAC (A,B,C) and unweighted UNIFRAC (D,E,F) results were generated and colored by depth (A,D), sediment source (B,E) or cellulose source (C,F). 100 rarefactions were conducted at a depth of 800 sequences per sample to estimate robustness of beta diversity patterns. Shading around each point represents interquartile range of that point's placement as calculated based on rarefied PCoA.</p

Winogradsky columns.

<p>Triplicate columns of each condition were prepared. Photographs were taken following sample collection. The teal plugs covering the holes drilled can be seen in some columns; the topmost plug is at the SWI. Column numbers are shown below the image.</p

Distribution of abundant and rare genera.

<p>Samples were categorized by depth or sediment source and the average abundance of each genus was calculated. Number of genera (percent) is shown.</p

Heatmaps of abundant taxa in Winogradsky columns.

<p>Relative abundance of the six most abundant phyla is shown for (A) Buxton Pond and (B) Eph's Pond, and of Proteobacteria classes in (C) Buxton Pond and (D) Eph's Pond. Samples are organized by column depth from top to bottom.</p