This research is the first culture-independent phylogenetic study of bacterial diversity in sediments of constructed wetlands for the bioremediation of acid mine drainage (AMD). AMD is a product of the biological and chemical oxidation of sulfides co-extracted with ores, resulting in elevated sulfate and heavy metal concentrations, and depressed pH. Traditional chemical approaches to the treatment of AMD are expensive and pose significant problems, so passive biological systems have been constructed in an attempt to exploit naturally occurring macro- and micro-biological processes to ameliorate AMD in a cost-effective manner. Constructed wetlands to treat AMD are poorly understood microbiologically, even though microorganisms mediate the most significant processes occurring in the sediment. It is recognised that the sulfate-reducing bacteria (SRBs) are an important metabolic group for AMD treatment, and since the concentration of electron donors in AMD limit the effectiveness of SRB in sediments, the presence of populations of cellulose degrading bacteria (CDB) in sediment microbial communities to supply the necessary organic electron donors to the SRB may be crucial.
A molecular approach was taken to assess the diversity of the wetland sediment dominated by species of the cattail Typha latifolia using 16S rRNA gene phylogeny. To achieve this, clone libraries of 16S rDNA were generated from sediment and rhizosphere samples taken from the constructed wetland. The results indicated a difference between the sediment and the rhizosphere, with a drop in the number of SRB in the rhizosphere. The presence of 16S rDNA clones affiliated to known groups of SRB and CDB indicated the presence of representatives of both of these functional groups in the wetland sediment. When analysed phylogenetically, some 16S rDNA clones were found to be related to species of known sulfate reducers such as the group containing Desulfosarcina variabilis and Desulfobacterium indolicum within the family Desulfobacteraceae of the δ- Proteobacteria and Desulfitobacterium sp. in the Low mol. % G+C Gram-positive bacteria. In addition, a significant number of 16S rDNA clones showed close relationships to described CDBs, including Clostridium termitidis, [Bacteroides] cellulosolvens, Termitobacter aceticus and Thermoanaerobacterium spp. However, many clones were not closely related to described species of SRB in the b-Proteobacteria and CDB in the Low mol. % G+C Gram Positive Bacteria and most likely represent novel phylogenetic lineages at •the genus level. One phylogenetic cluster represented by novel clones in the 'Desulfobacteraceae' was studied in detail.
The gene for a key enzyme in sulfate reduction, dissimilatory sulfite reductase (dsr) , was detected and amplified using degenerate PCR primers from sediment mixed community genomic DNA and cloned. Due to lateral gene transfer events in the evolutionary history of dsr, it was not possible to directly compare 16S rDNA and dsr phylogeny. However, dsr clones were found which are related to: 1) known sulfate reducers such as Desulfomonile tiedjei, Desulfoarculus baarsi, and a laterally-transferred dsr in Desulfobacula toluolica; 2) novel clone groups with no cultured representatives, and 3) novel lineages which did not contain cultured representatives or environmental clones.
PCR primers and 16S rRNA/rDNA probes were developed from the 16S rDNA sequences obtained from the clone library studies and were applied to detect SRBs in sediment samples including the novel clone group. Molecular techniques including fluorescence in-situ hybridization (FISH), PCR, and hybridization using DIG-labelled probes were used to study SRBs in both the sediment and rhizosphere samples.