## 4 Discussion  
From the aforementioned results, it becomes evident that, upon introduction of TOPW, a drastic shift in the prokaryotes and eukaryotes present takes place; the organisms naturally present in TOPW and its specific chemical composition seem to exert a major pressure on the bio-community dynamics [50]. Such kind of shifts in wastewater treatment plants have already been described for other environmental factors, such as temperature [24] or salinity [51]. The number of OTUs identified in all samples is very large (39,108) and the significant bacterial OTUs present above 0.1% in abundance increase in the acclimatized MBR (Day0 = 95, Day87 = 119) indicating a strong diverse microbial biodegradation community. All major OTUs present in the samples from the latter part of the operating period are present in the Day16 sample, while the dominant OTUs present in active sludge disappear or drastically decrease. The acclimatization process of the MBR is significantly faster than it was hypothesized. The increase of the bioreactor pH value, together with the increase of the MLVSS, are indicators of a successful acclimatization of the microbial bio-community in the MBR and the biodegradation of organic matter present in the wastewater. The stable pH value (pH = 8.5), after Day20, has been reported in previous studies of biological treatment of wastewater originating from olive processing [52] and can be attributed to the efficient biodegradation of the organic acids in the bioreactor that results in rise of the pH value. Moreover, the operating efficiency in terms of TOC and polyphenols removal is quite high, while the acclimatization phase is on-going; the removal ratios in 90% of the analyzed samples were over 90% and 85% for TOC and TPh, respectively, denoting a rather stable biodegradation process. Figure 9: Prokaryotic community taxonomic analysis of the metatranscriptomic data. Distribution of the top identified bacterial organisms at genus level, identified in the three time points for the metatranscriptome analysis. The scale in the y axis reflects the normalized relative abundance percentages (%). Black lines within each bar separates each genus into different species. From the prokaryote bio-community, _Thuuera_ and _Paracoccus_ spp. remained present throughout MBR operation and appear to be important for the bioremediation process. Further studies on these two species may provide further insights into TOPW treatment. _Thuera_ spp. have been recognized as specialists on lactate conversion, which is the main by-product of TOPW fermentation, as well as a wide variety of aromatic substrates under aerobic or denitfying conditions [48]. The aromatic ring cleavage ability may be also employed for polyphenolic substances degradation given the relevance in the chemical structure of some aromatic substances (like benzoate and its hydroxylated derivatives) and simple polyphenols. _Paracoccus_ spp. is capable of storing a variety of polyhydroxyalkanoates reserves of carbon and energy [53] as a response to feast/famine conditions. Microorganisms that possess such metabolic mechanisms (i.e., assimilation of internal carbon and energy pools, like glycogen and poly-P granules) take advantage of the feast/famine conditions of the MBR due to the periodic (once a day) feeding pattern; this is a characteristic indication of the significant effect that operating conditions have on the microbial community. RNA based metatranscriptomics analysis is believed to provide more precise information on community functions by identifying only expressed gene transcripts, actively metabolizing microbes, and thereby more accurately reflect the pool of enzymes that are active within the community [54]. Surprisingly, the relative abundance of expressed prokaryotic genes differed significantly from the abundance of microbial organisms enumerated by 16S rRNA. A handful of species (_Thuera_, _Paracoccus_ spp.) dominate the expression frequency, whereas numerous others maintain quite low levels of expression, usually lower than 1-2%.