Profiling in situ microbial community structure with an amplification microarray

Abstract

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO(3), but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Fig 1

Aggregate signal intensity (as a proportion of the total signal) for the amplification microarray (A) and hybridization microarray (B). Only probes that were common between the two microarray platforms are considered.

Fig 2

Microbial community dynamics in response to acetate and bicarbonate treatments. CD-11 sample 28-Oct was negative by amplification microarray, hybridization microarray, and qPCR, indicative of residual PCR inhibitors in the nucleic acid extract. MTBE, methyl tert-butyl ether. *, the Ferribacterium probe also has perfect sequence identity with Dechloromonas aromaticum and is therefore not in and of itself an absolute indicator of Ferribacterium presence.