## 3 Results
### 16S and 18S Amplicon Meta-Barcoding  
To assess the microbial community dynamics during acclimatization and operation, meta-barcoding analysis was performed for both prokaryotic and eukaryotic organisms, using the 16S rRNA and 18S rRNA genes, respectively, as taxonomic identification markers. Sequencing of the 16S rRNA gene resulted in 126,360, 140,229, 207,649 and 213,719 filtered reads for Day0, Day16, Day58 and Day87 time points, respectively (Table 2). For eukaryotes, the respective numbers were 147,664, 182,962, 169,154 and 151,733. In total, 39,108 and 3471 unique OTUs were obtained for bacteria and eukaryotes, respectively. The total number of identified bacterial OTUs per sample ranged from 12,597 (Day0) to 16,755 (Day87) (Table 2). However, the significant OTUs exceeding 0.1% in abundance in each sample time point were significantly less (Day0: 95 OTUs, Day16: 96 OTUs, Day58: 107 OTUs and Day87: 119 OTUs). Comparisons of the shared organisms between samples identified a rather swift transition from the active sludge population at Day0 to an alternative core microbial community present at later stages of MBR function. The percentage of shared OTUs between Day0 and Day16 was 10.4%, which further diminished to 1% when compared to Day58 and Day87, whereas 44% of OTUs were common in the later samples. The active sludge inoculum (Day0) was dominated by _Proteobacteria_ (68%) and _Firmicutes_ (11%). _Actinobacteria_ (6%), _Chloroflexi_ (4%), _Bacteroidetes_ (3%), and _Planctomycetes_ were the minor phyla of the community (Figure 6). _Alphaproteobacteria_ consisted of three OTUs of the order \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Amplicon** & **Sample** & **Raw Reads** & **Number of Sequencesafter OTU Picking** & **No OTUs** \\ \hline \multirow{4}{*}{16S} & Day0 & 221,672 & 126,360 & 12,597 \\  & Day16 & 218,040 & 140,229 & 13,890 \\  & Day58 & 290,839 & 207,649 & 15,357 \\  & Day87 & 291,227 & 213,719 & 16,755 \\ \hline \multirow{4}{*}{18S} & Day0 & 221,664 & 147,664 & 1472 \\  & Day16 & 269,715 & 182,962 & 876 \\ \cline{1-1}  & Day58 & 255,537 & 169,154 & 1038 \\ \cline{1-1}  & Day87 & 213,397 & 151,733 & 673 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Number of raw reads, sequences after operational taxonomic unit (OTU) picking and identified OTUs, for each timepoint, identified for 16S rRNA and 18S rRNA amplicon sequencing. Figure 5: Membrane temperature corrected (20 \({}^{\circ}\)C) permeability. Data obtained during MBR acclimatization period; the arrows indicate sample collection time points for metagenomic analysis; the shaded area indicates the time period when both CSW and TOPW were fed to the MBR, CSW, and TOPW were solely fed before and after this period, respectively. _Rhizobiales_ which amounted to 42% of the total OTUs of Day0. One of them, a _Methylocystis_ spp., is a known methanotroph [42]; its existence is attributed to the fact that the initial inoculum comes from a WWTP that treats sewage wastes (sludge) from municipal septic tanks where anaerobic conditions and methane formation prevails. These three OTUs were effectively eliminated from the later samples. _Betaproteobacteria_, comprising 18% of the Day0 sample were dominated (>99%) by a single genus, _Dechloromonas_ spp., a facultative aerobic, nitrate-reducing organism frequently encountered in WWT plants with a capacity to accumulate polyphosphate [43] (Figure 6). Its presence diminished in the MBR while another member of the _Rhodocytaceae_ family, _Thuera_ spp. present initially in scarce numbers, increased in abundance displacing _Dechloromonas_ spp. (Figure 6). _Frimicutes_, totaling 11% of Day0 active sludge, consisted of a single _Trichococcus_ spp., a filamentous facultative anaerobe, fermenting readily metabolizable substrates. Its population also declined to zero in the MBR (Day58, Day87), where aerobic conditions prevail (Figure 6). _Actinobacteria_ increased in the MBR to a significant percentage and number of species. The main genus at Day0, _Dietzia_, was replaced by several OTUs.