PNNL

Abstract

A 16S rRNA-targeted tunable bead array 2 was developed and applied in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface 4 sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without employing a polymerase chain 6 reaction step. Bead array validation studies with total RNA derived from 26 isolates indicate that the behavior and response of the 16S rRNA-targeted oligonucleotide probes 8 could not be predicted based upon primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the relative abundance of 10 one organism (or rRNA) to another. Nevertheless, microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were 12 consistent with previous data acquired at the site, where indigenous sulfate and iron reducing bacteria and Desulfotomaculum near neighbors were the most responsive to a 14 change in injected acetate concentrations. Interpreting bead array data was best accomplished by analyzing the relative change in probe response over spatially and 16 temporally related samples, and by only considering the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the data 18 interpretation in this manner and placing it within the context of supporting geochemical and microbiological analyses, we conclude that ecologically relevant and meaningful 20 information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even amidst current analytical uncertainty surrounding individual 22 probe behavior on tunable bead arrays.