Application of molecular techniques to elucidate the influence of cellulosic waste on the bacterial community structure at a simulated low-level-radioactive-waste site

Abstract

Low-level-radioactive-waste (low-level-waste) sites, including those at various U.S. Department of Energy sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a nonradioactive model low-level-waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more operational taxonomic units, and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the fill and fill-waste interface (FW) layers and greater in the wood waste and waste-clay interface layers. Principal-coordinate analysis and lineage-specific analysis determined that the Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose-degrading microorganisms suggest that the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.

FIG. 1.

Schematic of the nonradioactive CTPS near the LLW site at the INL where soil samples were obtained. Brackets indicate sampling points. F, fill; FW, fill-waste interface; WW, wood waste; WC, waste-clay interface.

FIG. 2.

The bacterial community viewed at the phylum level with depth at the CTPS. (A) Percent abundance of each phylum as determined by clone library analysis with the total number of clones for that layer listed at the top of each bar. (B) Number of unique OTUs identified within each phylum based on clone library (CL) and PhyloChip (PC) analyses. F, fill; FW, fill-waste interface; WW, wood waste; WC, waste-clay interface.

FIG. 3.

PCoA of the (A) combined clone libraries, (B) combined PhyloChip data, and (C) combined clone library and PhyloChip data. A 97% identity cutoff was used to remove replicate sequences from the clone libraries before analysis. F, fill; FW, fill-waste interface; WW, wood waste; WC, waste-clay interface.

FIG. 4.

(A) Acidimicrobiaceae and (B) Streptomycetaceae families within the Actinobacteria phylum and (C) Flexibacteraceae and (D) KSA unclassified families within the Bacteroidetes phylum that had significant changes with depth as viewed by PhyloChip and clone library analyses. PhyloChip results are presented as a presence (black)-absence (gray) heat map for each OTU detected within the family. Each row marked by a dot on the left represents a unique OTU. An OTU was determined to be present in a soil layer if the pf value was above or equal to 0.92 for both PhyloChips. Clone abundance of each family is reported as the percentage of the total clones detected per soil layer. Quantitative PCR was performed using family-specific primers for amplification of the 16S rRNA gene. F, fill; FW, fill-waste interface; WW, wood waste; WC, waste-clay interface.

FIG. 5.

Focus group comparisons of the Actinobacteria phylum. Families with a significant decrease or increase in the clone number between at least two layers (e.g., significant change between the F and FW layers) were categorized as either reported and implied cellulose degraders (families that were previously known to be cellulose degraders or cellobiose utilizers or have been suggested to be potential cellulose degraders) or non-cellulose degraders (families that have not been shown in the literature to degrade cellulose or utilize cellobiose nor suggested to be able to). These two groups were then compared based on (A) clone abundance and the number of OTUs detected by (B) clone library and (C) PhyloChip analyses. F, fill; FW, fill-waste interface; WW, wood waste; WC, waste-clay interface.