Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation

Abstract

Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.

FIG. 1.

Plots show DNA concentration (A) and microbial activity (dehydrogenase) (B) in soil samples taken from Area 2 soil before column packing and during net U reduction and oxidation phases. Bars show means ± standard errors (n = 3).

FIG. 2.

PCA of 16S T-RFLP profiles showing bacterial community divergence from original FRC Area 2 soil prior to column packing and through net uranium reduction and reoxidation phases.

FIG. 3.

Rarefaction analysis of clone library coverage of microbial diversity in uranium-contaminated Area 2 soil samples. Two sequence homology levels are shown: 100% represents the coverage estimate when clones are placed in groups using this homology cutoff and 99% represents the coverage estimate based on clones grouped using a 99% sequence homology cutoff, which corresponds to the typical divergence of sequences within array-defined OTUs.

FIG. 4.

Neighbor-joining tree showing phylogenetic position of 16S rRNA gene clones generated from Area 2 soil prior to column packing. Sequences were inserted by parsimony into a tree containing all bacteria and archaea in the “greengenes” database (as of 3 May 2005). Abbreviations are as follows: β-proteo, Betaproteobacteria; γ-proteo, Gammaproteobacteria; δ, Deltaproteobacteria; G, Gemmatimonadetes; Bact, Bacteroidetes; Ver, Verrucomicrobia; P, Planctomycetes; W, candidate division WS3; D, candidate division DSS1; Acido, Acidobacteria; Chlorof, Chloroflexi; T, candidate division TM7. Values in parentheses indicate numbers of clones in each group. Methanobacterium subterraneum strain C2BIS was used to root the tree. The scale bar represents 10 inferred nucleotide changes per 100 nucleotides analyzed.

FIG. 5.

Chao1 richness estimates of Area 2 soil bacterial communities based on clone library sampling. Richness estimates were made using a 99% sequence homology group classification equivalent to array-defined OTUs. The dashed gray line denotes the mean number of OTUs detected by array in Area 2 samples. The dashed black line denotes the number of OTUs detected by clone sequence analysis in Area 2 samples.

FIG. 6.

Heatmap and dendrograms showing the response of 100 bacterial subfamilies (shown on y axis) exhibiting the highest standard deviation between samples (shown on x axis) taken pre-lactate stimulation (Area2.1, Area2.2, and Area2.3), during net uranium reduction (Red1, Red2, and Red3), and during net uranium reoxidation (Ox1, Ox2, and Ox3). The color gradient from green to red represents increasing array hybridization intensity. Five main response groups were detected, and the average intensity (HybScore) of the cluster group response is presented in line plots to the right of the heatmap. Error bars represent standard errors in HybScore differences between all subfamilies in a cluster.