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TECHNICAL REPORTS

Wetlands and Aquatic Processes

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

^* Corresponding author (dick.5{at}osu.edu)

Received for publication August 24, 2005. Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at <0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant. Heterotrophs in the Acidiphilium genus totaled 20% of the bacterial population. Leptospirillum ferrooxidans was below the level of detection in the bacterial community. The results from the FISH technique from this field study are consistent with results from other experiments involving enumeration by most probable number, dot-blot hybridization, and denaturing gradient gel electrophoresis analyses and with the geochemistry of the site.

Abbreviations: AMD, acid mine drainage • DAPI, 4',6-diamidino-2-phenylindole • DGGE, denaturing gradient gel electrophoresis • dUTP, 2'-deoxyuridine-5'-triphosphate • EDTA, ethylenediaminetetraacetic acid • FISH, fluorescence in situ hybridization • MPN, most probable number • PBS, phosphate buffered saline • PCR, polymerase chain reaction • SDS, sodium dodecyl sulfate • SSC, sodium chloride–sodium citrate • TE, trishydroxymethylaminomethane–EDTA