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Microbial Populations Identified by Fluorescence In Situ Hybridization in a Constructed Wetland Treating Acid Coal Mine Drainage

^a Environmental Science Graduate Program, The Ohio State University, Columbus, OH 43210. Present address: National Science and Technology Development Agency, 111 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
^b Environmental Science Graduate Program and School of Environment and Natural Resources-Soil Science, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691
^c Environmental Science Graduate Program and Department of Microbiology, The Ohio State University, Columbus, OH 43210

View larger version (27K): [in a new window]   Fig. 1. Experimental flowchart of this study.

View larger version (66K): [in a new window]   Fig. 2. Photomicrographs of iron precipitate samples. (A) Stained with 4',6-diamidino-2-phenylindole (DAPI) (top) and hybridized with universal probe Univ-1392 (bottom). (B) stained with DAPI (top) and hybridized with eukaryotic universal probe Euk 502 (bottom). (C) Stained with DAPI (top) and hybridized with bacterial universal probe EUB338 (bottom). Scale bar marker: 10 µm.

View larger version (69K): [in a new window]   Fig. 3. Photomicrographs of cells in Fe precipitate samples. (A) Stained with 4',6-diamidino-2-phenylindole (DAPI) (top) and hybridized with probe TF539 for A. ferrooxidans (bottom). (B) Stained with DAPI (top) and hybridized with probe Acdp821 for Acidiphilium spp. (bottom). Scale bar marker: 5 µm.

View larger version (111K): [in a new window]   Fig. 4. Dot-blot hybridization of DNA extracted from December samples from Cell 1 (A and B) and Cell 4 (C and D). Amplified 16S rDNA from A. ferrooxidans (A1 and C1), A. thiooxidans (A2 and C2), Archaeoglobus fulgidus (B1 and D1), and Ferroplasma acidarmanus (B2 and D2) was hybridized by using fluorescein-labeled rDNA from Fe precipitates.

View larger version (17K): [in a new window]   Fig. 5. Culturability of acidophilic bacteria in oxic wetland sediments. The most probable number (MPN) counts of acidophilic Fe and S oxidizers, and glucose-oxidizing heterotrophs were compared to fluorescence in situ hybridization (FISH) counts obtained with probes TF539 (A. ferrooxidans), Thio820 (A. thiooxidans), and Acdp821 (Acidiphilium spp.) that were taken to be 100%. The corresponding FISH counts are listed in Table 2.