## 3 Results
### 16S and 18S Amplicon Meta-Barcoding
At Day16, seven major actinobacterial OTUs were identified collectively amounting to 28% of the microbial community. Two _Tessaracoccus_ spp. accounted for one third of them. Members of the genus have been associated with enhanced biological phosphate removal and accumulate polyphosphate granules [44]. Another actinobacterial species which became dominant in the MBR was _Nakamurella_ spp. (Figure 6). It can utilize numerous sugars and amino acids as carbon sources and accumulates glycogen possibly competing with phosphate accumulating bacteria. Another introduced organism from the TOPW feed which likely affects the community balance was _Bdellovibrio_ spp., a _Deltaproteobacteria_ organism parasitizing on other Gram-negative bacteria, which could explain the decrease in their abundance in the MBR [45]. The nitrate reducing organisms _Paracoccus_ spp. and _Thuera_ spp. which increased in abundance were complemented with the nitrite oxidizing bacterium (NOB) _Nitrospira_ spp. absent from Day0, but present at significant levels subsequently. _Nitrospira_ is a globally distributed genus and represents the most diverse known group of NOB. Aside from the capacity to use nitrite as energy source, certain species of _Nitrospira_ possess the capacity for complete ammonia oxidation [46]. Figure 6: Metagenomic analysis of prokaryotic community. Distribution of (**a**) the major bacterial phyla, identified at four timepoints during MBR operation. The scale in the y axis reflects the normalized relative abundance percentages (%). Black lines within each bar separates each Phylum into lower taxonomic levels and (**b**) the relative abundances of the top 25 bacterial genera, identified in the four timepoints visualized through heatmap. Analysis of the eukaryotic load in the MBR community by 18S rRNA identified three major phyla, _Opisthokonta_, _SAR_, and _Archaeplastida_, accounting for most organisms and a pool of unclassified organisms ranging from 11% at Day0 to single digit percentages for the other time points (Figure 7). The main phylum at Day0, _Opisthokonta_, includes the true fungi and their protist relatives, as well as the animals and their protist relatives [47]. At Day0 sludge sample, 73% of the reads are _Opisthokonta_, and they remain substantial at later time points (Day16 = 45%, Day58 = 32%, Day87 = 57%) (Figure 7). True fungi were 47% of all reads at Day0, and their abundance decreases at later samples but remains at high levels (Day16 = 27%, Day58 = 11%, Day86 = 19%). _Ascomycota_ (20%), _Basidiumycota_ (15%), and _LKM11_ (9%) a _Cryptomycota_, are the dominant genera (Figure 7). During MBR acclimatization _Ascomycota_ and _Basidiumycota_ diminish in abundance (OTUs 00008, 00013, 00015, 00016, 00019, 00021) and a single OTU from each, remains in significant numbers. In the _LKM11_ clade, the single dominant OTU00009 at Day0 decreased dramatically, while a second OTU00004 absent at Day0 expanded to account for 18% of the total reads at Day16 and subsequently stabilizing at lower levels. The metazoan members of _Opisthokonta_ belong to genus _Bilateria_, with a single OTU00003 accounting for 99% of the genus. It is present in all samples in stable abundance (12-19%). The phylum _SAR_ clusters taxonomically three clades, _Stranenopiles_, _Alveolates_, and _Rhizaria_[47]. The _SAR_ phylum expanded from 14% of the reads at Day0 to 50% at Day16, 64% at Day58 and 37% at Day87. _Rhizaria_ account for 99% of _SAR_ organisms. They are a group composed of numerous unicellular species mostly amoeboid in appearance. All OTUs were _Cerozoa_. Two OTUs dominate, a _Cryomonadida_ genus present in all samples, with a peak at Day16 (Day0 = 6%, Day16 = 48%, Day58 = 8%, Day87 = 9%) and an unclassified genus absent at Day0 which expanded to 56% at Day58 and 31% at Day87.