Water contamination by toxic and relatively recalcitrant cyclic ether compounds, particularly 1,4-dioxane (dioxane) and tetrahydrofuran (THF), is a growing concern. However, our knowledge about dioxane degraders is limiting, and traditional isolation techniques (e.g., enrichment and serial dilution for separating colonies on plates) are challenged. In this study, first we developed oligonucleotide probe Pse631 targeting the 16S rRNA of Pseudonocardia members with high coverage and specificity. In combination with flow cytometry and cell sorting, in situ hybridization with this probe enables sensitive and specific detection of Pseudonocardia cells in mixed cultures and enriched environmental samples without significant false positives, using Escherichia coli, Bacillus subtilis, and Mycobacterium spp. as negative controls. The functional gene analysis by quantitative PCR confirmed effective separation of Pseudonocardia cells from the mixed consortium, and also linked metabolic potential with 16S-based identification of the specific sub-population. Then we consummated our approach by designing some functional gene probes targeting the α subunits of tetrahydrofuran/dioxane monooxygenases (thmA). With this probe, we can successfully label known 1,4-dioxane degraders (e.g., Psedonocardia dioxanivorans CB1190), and also isolate putative degraders from two contaminated sites. Phyla Actinobacteria, Proteobacteria and Bacteroidetes were noticed to be dominant in sorted cells originated from the both contaminated sites. A group of Rhizobiales, (up to 94.2% in sorted cells) including Aminobacter, Nordella, Mesorhizobium, Bradyrhizobium and Ensifer, were present, and this suggests applicability of phytoremediation in dioxane-contaminated sites. This study described a culture-independent high-throughput molecular approach to isolate microbial populations of interest from complex microbial communities and that these sub-populations can be used for subsequent genetic tests to estimate their performance in ecological niches.

Discovering and sorting potential 1,4-dioxane degrading bacteria by fluorescence in situ hybridization and flow cytometry using phylogenetic or functional oligonucleotide probes

256th National Meeting (2018)

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