Exposure of Soil Microbial Communities to Chromium and Arsenic Alters Their Diversity and Structure

Extensive use of chromium (Cr) and arsenic (As) based preservatives from the leather tanning industry in Pakistan has had a deleterious effect on the soils surrounding production facilities. Bacteria have been shown to be an active component in the geochemical cycling of both Cr and As, but it is unknown how these compounds affect microbial community composition or the prevalence and form of metal resistance. Therefore, we sought to understand the effects that long-term exposure to As and Cr had on the diversity and structure of soil microbial communities. Soils from three spatially isolated tanning facilities in the Punjab province of Pakistan were analyzed. The structure, diversity and abundance of microbial 16S rRNA genes were highly influenced by the concentration and presence of hexavalent chromium (Cr (VI)) and arsenic. When compared to control soils, contaminated soils were dominated by Proteobacteria while Actinobacteria and Acidobacteria (which are generally abundant in pristine soils) were minor components of the bacterial community. Shifts in community composition were significant and revealed that Cr (VI)-containing soils were more similar to each other than to As contaminated soils lacking Cr (VI). Diversity of the arsenic resistance genes, arsB and ACR3 were also determined. Results showed that ACR3 becomes less diverse as arsenic concentrations increase with a single OTU dominating at the highest concentration. Chronic exposure to either Cr or As not only alters the composition of the soil bacterial community in general, but affects the arsenic resistant individuals in different ways

Alpha-diversity analysis using rarefaction (A) and phylogenetic diversity (B) of contaminated and control soils.

<p>Each soil is represented by color and pattern. Rarefaction analysis focuses on discovery of novel species level (97%) Operational Taxonomic Units (OTUs) while phylogenetic diversity focuses on the discovery of novel phylogenetic branches.</p

Pyrosequencing results, diversity estimates, and 16S rRNA gene copy abundance for each sampling site.

1<p>Diversity Estimators Abbreviations: Abundance-based Coverage Estimator (ACE), Chao’s species richness estimator, and Shannon-Weiner Index.</p>2<p>Species level, 97% similarity threshold used to define Operational Taxonomic Units (OTUs).</p

Effect of metal contamination, pH and soil organic matter on diversity and evenness metrics of total soil communities¹.

1<p>Significance was assessed using a two-tailed t-test (n = 9) and p-values are represented by: * ≤0.05, ** ≤0.01, and *** ≤0.001.</p>2<p>Abbreviations: Abundance-based Coverage Estimator (ACE), Chao’s species richness estimator, Shannon-Weiner Index, Faith’s Phylogenetic Diversity, and ns, not significantly different.</p

Effect of As and Cr(VI) contamination on β-diversity of the microbial communities from control and contaminated sites.

<p>The first two coordinate axes from a principal coordinate analysis (PCoA) were plotted using the unweighted (A) and weighted (B) Unifrac algorithms. Colors represent paired control and contaminated sites.</p

Phylogeny of <i>ACR</i>3 OTUs recovered from contaminated soil clone libraries.

<p>Neighbor-joining tree showing relationships between the <i>ACR3</i> clone library sequences, ACR3 genes from the As-resistant isolates, and closely related sequences from the NCBI database. <i>ACR3</i> sequences generated in this study are in bold and the inlaid graph shows the distribution of OTUs with more than 1 sequence.</p

Diversity of <i>ACR3</i> genes versus increasing arsenic concentrations.

<p>Graph shows the Shannon Weiner diversity indices for the clone libraries and T-RFLP results of the <i>ACR3</i> gene, as well as the arsenic concentration of the three sample sites. The figure illustrates the diversity trends observed for <i>ACR3</i> across the three sites, and how they relate to the most dominant OTU in the library. Shown are the diversity values for all three sites from Clone Library data, from T-RFLP data, and the abundance values for the most dominant OTU, ACR3PAK1.</p

Phylogeny of <i>arsB</i> OTUs recovered from contaminated clone libraries.

<p>Neighbor-joining tree showing relationships between the <i>arsB</i> clone library sequences, <i>arsB</i> genes from As-resistant isolates, and closely related sequences from the NCBI database. <i>arsB</i> sequences generated in this study are in bold and the inlaid graph shows the distribution of OTUs with more than 1 sequence.</p

Location and key geochemical data of contaminated and control study sites.

<p>Additional soil and chemical analyses are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040059#pone.0040059.s001" target="_blank">Table S1</a>.</p>1<p>BD: Below detection. Detection limit for chromium is 0.004 mg kg⁻¹ and 0.008 mg kg⁻¹ for arsenic.</p