## 3 Results and Discussion
### Microbiological Diversity  
The diversity and abundance of microbiological species in bottom sediment samples, collected at a depth of 8 cm, were analyzed using 165 rRNA next-generation sequencing. The sequences were grouped into operational taxonomic units (OTUs) based on their similarities. The results were categorized into dominant and other taxa, with the dominant taxa having an abundance of \(\geq\)1%, and the other taxa having an abundance of <1%. Notably, the other taxa constituted 44.99%, 38.57%, and 37.33% of the total OTUs detected at S1, S2, and S3, respectively. Although these taxa may be present in small numbers individually, their collective presence indicates a high level of microbial diversity at all three sites. Figure 7 shows that the dominant taxa included 21, 27, and 24 microbial families at S1, S2, and S3, respectively. Among the dominant families, those within the phylum _Proteobacteria_ (alpha, beta, and gamma) were the most prevalent. Other significant phyla included _Acidobacteria_, _Firmicutes_, and _Thermodesulfobacteria_. A previous study on the Zaalklapspruit wetland demonstrated a notable shift in the relative abundance and bacterial diversity within the bacterial consortium following ecological engineering interventions. Although _Proteobacteria_ remained the dominant phylum, its relative abundance decreased, while _Firmicutes_ significantly increased [8]. It is important to note that this earlier study focused on the water column bacterial consortium, which might account for the differences in the abundance percentages when compared to the sediment samples analyzed in the current study. Several studies on natural and constructed wetlands receiving AMD have highlighted the abundance of these taxa in sediment samples, underscoring the crucial role of microbially mediated activities in transforming metallic and sulfur compounds during AMD remediation [84; 85; 86]. This study identified thirteen comparable families as dominant taxa in the bottom sediment across S1, S2, and S3, reflecting the consistent microbial community structure throughout the wetland. The most prominent families included the following:  
1. _Heliobacteraceae_ > _Hydrogenophilaceae_ > _Rhodospirillaceae_ > _Callinocellaceae_ > _Xanthomonadaceae_. 2. _Acidobacteriaceae_ > _Gallinocellaceae_ > _Xanthomonadaceae_ > _Heliobacteraceae_ > _Hydrogenophilaceae_. 3. _Heliobacteraceae_ > _Gallinocellaceae_ > _Xanthomonadaceae_ > _Hydrogenophilaceae_ > _Rhodospirillaceae_. Figure 7: Distribution of the most dominant microbiological families (relative abundance > 1%) in the bottom sediment of each site. _Helicobacteraceae_ accounted for 19.98%, 6.14%, and 23.53% of the OTUs in the bottom sediment at S1, S2, and S3, respectively. This family, part of the phylum _Epsilonproteobacteria_, thrives at oxic-anoxic interfaces in sulfur-rich environments [87]. Studies have linked _Helicobacteraceae_ with mine effluents high in heavy metals and Fe concentrations, indicating their dominance in metal-polluted aquatic systems [88; 89; 90]. In some cases, they represent up to 65% of the microbial community in such environments. _Hydrogenophilaceae_, another significant family observed in this study, are strict autotrophic or facultative anaerobic sulfur oxidizers, playing a crucial role in sulfur utilization in wastewater treatment systems [91]. The family _Xanthomonadaceae_ constituted 6.07%, 7.44%, and 6.80% of the OTUs in S1, S2, and S3, respectively. Belonging to the phylum _Gammaproteobacteria_, _Xanthomonadaceae_ includes iron-reducing bacteria that oxidize Fe(II) for growth and are commonly found in AMD-polluted water bodies [84; 92]. Ref. [92] found that _Xanthomonadaceae_ represented approximately 51% of the detected _Gammaproteobacteria_ in sediments from AMD-induced iron mounds, highlighting their preference for Fe-rich environments. _Gallionelaceae_, also associated with Fe(II)-rich sediments and iron oxidation, showed a relative abundance of 10.47% at S3, where Fe\({}_{2}\)O\({}_{3}\) was prevalent in the bottom sediment (Table 3).