Effect of hydraulic retention time on microbial community structure in wastewater treatment electro‐bioreactors

Abstract The impact of hydraulic retention time (HRT) on the performance and microbial community structure of control and electro‐bioreactors was investigated. Control bioreactors and electro‐bioreactors were operated at HRT ranging between 6 and 75 hr. The total bacterial counts in addition to the removal efficiency of NH 4 +–N, sCOD, and PO 4 3−–P was assessed in all the reactors tested. In addition, Illumina sequencing was performed to determine the microbial communities that developed in these reactors under each HRT condition. Phylogenetic analysis showed that Proteobacteria and Bacteroidetes were the dominant phyla in those reactors. In addition, Nitrospira sp. and Pseudomonas sp. were found to be present in electro‐bioreactors with higher relative abundance than in control bioreactors. The results presented here are the first to determine what different microbial communities in wastewater electro‐bioreactors due to the application of an electric current under different HRTs.


| INTRODUCTION
Numerous wastewater treatment processes and techniques have been utilized to reduce water pollution and to improve drinking water quality. Understanding the behavior of microbial communities in biological processes has been recently the focus of many research studies.
The integration of electrochemical processes into membrane bioreactors (MBRs) combines biodegradation, electrochemical and membrane filtration processes into one system achieving high effluent quality as compared to conventional MBRs and activated sludge processes (Ensano et al., 2016). New and cheaper developments have suggested that the continuous and intermittent application of a direct current (DC) field has proven to enhance membrane filterability and is similarly effective in controlling membrane fouling (Akamatsu, Lu, Sugawara, & Nakao, 2010;Akamatsu et al., 2012;Liu, Liu, Gao, & Yang, 2012;Liu et al., 2014). The intermittent application also minimizes the direct exposure of bacteria to the electric field, potentially reducing the negative effects to the microbial community (Akamatsu et al., 2010;Bani-Melhem & Elektorowicz, 2010;Hasan, Elektorowicz, & Oleszkiewicz, 2014;Liu et al., 2012). We have previously reported that the application of DC at current densities of 5 and 10 Am −2 in electro-bioreactors led to enhancement in bioreactor performance as well as an increase in total bacterial counts and an apparent change in the microbial community structure (Zeyoudi et al., 2015). The performance and microbial community structure in an electro-bioreactor can be influenced by many operating parameters such as nutrient content, anoxic/aerobic phase fraction, salinity, solid retention time (SRT) and hydraulic retention time (HRT) (Fontenot, Bonvillain, Kilgen, & Boopathy, 2007). Among the above-mentioned parameters, HRT is regarded as one of the important operating parameters affecting the performance and microbial community of a bioreactor (Wang, Peng, & Stephenson, 2009). The effects of HRT and sludge properties (SRT and MLSS "Mixed Liquor Suspended Solids") in wastewater treatment using electrically enhanced MBR were previously explored by our group, where it was shown that increasing HRT is correlated with a reduction in COD, nitrogen and phosphorous content due to more exposure time of reactor content to biodegradation and electrocoagulation (Giwa & Hasan, 2015). Other studies reported the effects of HRT on the performance and microbial community of laboratory up flow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing trichloroethylene (TCE) (Zhang, Wang, Hu, & Li, 2015). Their results showed that the percentages of bacterial groups in each sample varied depending on the HRTs at different taxonomic levels where the potential function of dominant genera also showed and revealed the whole bacterial evolution of the biodegradation of TCE. High-throughput sequencing technologies have significantly improved researchers' ability to investigate microbial communities in various municipal and industrial WWTPs (Ibarbalz, Figuerola, & Erijman, 2013;Ma et al., 2015;McLellan, Huse, Mueller-Spitz, Andreishcheva, & Sogin, 2010;Roesch et al., 2007;Zhang et al., 2012;Zhang et al., 2015). Indeed, Illumina MiSeq has been successfully used to study various environmental and industrial systems in recent years (Caporaso et al., 2012;Gibson et al., 2014;Li et al., 2014;Liang et al., 2014). In this study, activated sludge samples were collected from the MBR plant at Masdar city and were analyzed using Illumina MiSeq after being used as inoculum in batch electro-bioreactors operated under different conditions for twenty-four hours. The sequencing data was analyzed using Quantitative Insights Into Microbial Ecology (QIIME ™ ) in order to elucidate alpha (α) and beta (β) diversity present in the different test reactors. Our data is the first to investigate the effect of HRT on the performance and microbial community structure of wastewater electro-bioreactors. The major objectives of this study were (a) to investigate the effect of HRT on the performance of bioreactor and electro-bioreactor; (b) to illustrate the effect of HRT on microbial community structure and function; (c) to differentiate between the effects of short HRT and long HRT linking the performance and functional bacterial groups in both bioreactors and electro-bioreactors.

| Electro-bioreactor experimental design
The experiments in this research study were conducted to evaluate the microbial community under current density of 3 Am −2 and HRT of 6, 10, 16, 24, 50 and 75 hr. The purpose of this design was to determine the operating conditions that favor an optimal effluent quality and microbial community. The selected range of 3 Am −2 was according to a previous study by our group (Zeyoudi et al., 2015), whereas the range of selected HRTs was in accordance with industrial scale MBR wastewater treatment plants (Tchobanoglous, Metcalf & Eddy, Burton, & Stensel, 2003). All bioreactors were fed with synthetic wastewater (0.2% Glucose, 1.5 mmol/L ammonium sulfate, 270 μmol/L potassium phosphate, 160 μmol/L magnesium sulfate, 20 μmol/L manganese sulfate, 1.47 μmol/L iron (III) chloride, 20 μmol/L calcium chloride, 330 μmol/L potassium chloride, 300 μmol/L sodium bicarbonate). Fresh activated sludge was collected from Masdar city's MBR wastewater treatment plant (Abu Dhabi -UAE) and used immediately to avoid any changes in its physiochemical and microbiological characteristics. The system used for this study was aerobic batch electro-bioreactors containing sludge and synthetic wastewater prepared in the laboratory. Each bioreactor was operated under a different HRT, which was calculated as per the effective volume of the reactor (V) and the feed (i.e., synthetic wastewater) flow rate (Q) according to HRT = V/Q. For example, 6 hr HRT was calculated via adding 1200 ml of wastewater per day to 300 ml of sludge sample in a batch mode. The same method was followed to adjust the other HRTs. A total of 12 (as shown in Figure 1a) bio-and electro-bioreactors were operated in parallel to ensure consistency in all experiments though which the same sludge having the same physiochemical and biological characteristics was used. The electrodes used in all experiments consisted of rectangular sheets of perforated aluminum with 75% opening as the anode, and stainless steel as the cathode spaced 5 cm apart. The effective surface area was calculated in each operating condition depending on the volume of the sample, by multiplying the width by the immersed length of the anode in the bioreactor. Aeration was provided via one small ceramic ball air stone diffusers (2-inch diameter) placed in each reactor, connected to air pumps placed at the bottom in order to provide oxygen necessary (>2 mg L −1 ) for aerobic microbial growth and to ensure homogenous mixing, thus no loss due to evaporation was assumed to occur. A reference control bioreactor (0 Am −2 ) was used in all conditions and had no electrodes.

| Synthetic wastewater and sludge characteristics
Synthetic wastewater and sludge dissolved oxygen (DO in mg L −1 ), pH, temperature (T in °C) and electrical conductivity (EC in μS cm −1 ) were analyzed using a HACH HQ40d single-input multi-meter probe At the end of each experiment, oxygen uptake rate (OUR) was measured after cutting off aeration in all bioreactors through which the DO probe (Hach HQ40d) was immersed in the sludge and DO depletion was monitored by taking a reading every minute over a period of 15 min. The slope of the DO vs time plot represents the OUR (in mgO 2 L −1 hr −1 ).

| Sampling and bacterial counts
A 10 ml sample from each reactor after 24 hr was collected from the zone between the electrodes and analyzed for bacterial counts (Zeyoudi et al., 2015). The total bacterial count (TBC), determined using the plate count method (Zeyoudi et al., 2015), was done in duplicate and an average value was recorded. Briefly, 0.1 ml was taken from each sample and serially diluted from 10 −1 to 10 −8 , followed by spreading 0.1 ml from each dilution on a Luria Broth plate (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar) and incubating at 37°C for 24 hr. The TBC from each reactor was calculated and reported as colony-forming units per milliliter (CFUmL −1 ). Results were expressed as the mean ± standard deviation. An analysis of variance was used to test the significance of the results with p < .05 determined a priori to be statistically significant. to our laboratory were analyzed by QIIME ™ (version 1.9.1), using published bioinformatics pipelines (Kuczynski et al., 2011). Before generating any figures, we filtered the QIIME ™ produced biom files by removing all unassigned operational taxonomic units (OTU's) and any OTU that did not at least have 5 counts in at least one of the samples tested. (α) and (β) diversity analysis were conducted to assess the microbial diversity in the serially passaged bioreactor  and electro-bioreactors (Jost, 2007). A phylogeny-based-weighted UniFrac distance analysis (Ju & Zhang, 2014) was used to compare between bacterial communities while BIO-ENV trend and Spearman's rank correlation analysis was performed to investigate for electric field and operational parameters that is correlated with bacterial community diversity.

| Reactor performance and physiochemical parameters under different HRT
In order to evaluate the effect of varying HRT on bioreactor performance, the different wastewater bioreactors tested were operated at HRTs of 6, 10, 16, 24, 50 and 75 hr for 24 hr. The

| Effect of HRT on total bacterial counts (TBC in CFU mL −1 )
Bacteria form an essential component of the microbial community representing the activated sludge. Their total count fluctuations can reflect the efficiency of the sludge to process the organics found in wastewaters. It is well understood that using the plate count method to enumerate microbes in environmental samples vastly underestimates numbers because the vast majority of microbes do not grow in laboratory cultures (Madigan et al., 2014). Despite this, it has been extensively used to compare samples and represents one way of measuring how a variable affects viability of microbes in a sample. The plate count method was used to determine bacterial counts in the various bioreactor configurations we tested.
Bacterial counts in the control bioreactors increased as HRT was increased and peaked at 11.8 × 10 6 CFU mL −1 at HRT of 50 hr, then dropped severely at HRT of 75 hr (2.5 ± 0.1 × 10 6 CFU mL −1 ).
( Figure 2e). This means that dilution, as the main consequence of high hydraulic load, has affected the microbial community size which is the decrease in sludge biomass and disaggregation of larger flocs. Interestingly, bacterial counts in electro-bioreactors were significantly increased compared to the controls at HRT's of 24, 50 and 75 hr (68.3 ± 0.4 × 10 6 vs 3.5 ± 0.1 × 10 6 , 23.9 ± 0.1 × 10 6 vs 11.8 ± 0.1 × 10 6 , and 3.6 ± 0.2 × 10 6 vs 2.5 ± 0.1 × 10 6 CFU mL −1 , respectively). The bacterial counts in control and electro-bioreactors were similar at HRT's of 6, 10 and 16 hr. This means that an electric T A B L E 2 Operational parameters of control bioreactors samples (C-6, C-10, C-16, C-24, C-50, and C-75) and electro-bioreactors samples (E-6, E-10, E-16, E-24, E-50, and E-75) field at current density of 3 Am −2 stimulated bacterial growth at higher HRT, which we have previously reported (Zeyoudi et al., 2015), but not at lower HRT's. These findings assume the high removal efficiency of sCOD, PO 4 3− -P and NH 4 + -N at different HRT tested could be due to electrokinetic process taken together with the high density of bacterial community present in the electrobioreactors (Henze, 2008).

| Overall bacterial diversity in control bioreactors and electro-bioreactors
Microbial community structure and function influences the performance of wastewater reactors, which in turn has an impact on the treated water quality (Henze, 2008;Tchobanoglous et al., 2003).
To compare the bacterial communities in bioreactors and electrobioreactors subjected to different HRT conditions, we performed (α) diversity analysis on each sample and (β) diversity analysis across the samples. α diversity expresses the diversity of a population within a system; a community will have a higher α diversity when there is a higher number of unrelated species within the same sample (Jost, 2007). α diversity was assessed, using the Chao1 index and Phylogenetic Diversity To investigate what physiochemical and operational parameters best correlated with the variability in bacterial abundance and diversity from each sample, A BIO-ENV trend and Spearman's rank correlation analysis (using QIIME v 1.9.0) was performed (that is β-diversity). This type of analysis calculates a bacterial community difference distance matrix and compares to Euclidian distance matrixes for each of the other mea-  50, and 75 hr on the right side. Therefore, it can be said the quite distinct microbial communities develop when short (6, 10, 16 hr) vs long (24, 50, 75 hr) HRT are used in the absence and presence of an electric current.
Indeed, it has previously been reported that microbial communities in various types of bioreactors are dependent on the HRT used (Klimiuk & Kulikowska, 2006;Zhang et al., 2015). In Figure 3e, we generated a PCoA plot to compare microbial community samples in the control bioreactor vs the electro-bioreactor running at the same HRT. The plot shows that microbial communities in control and electro-bioreactors are different at low HRTs (6, 10, and 16 hr) and converge to almost identical communities at an HRT of 24 hr, then diverge again at the longer HRT of 50 and 75 hr (Figure 3e).

| Phylogeny and abundance of overall bacterial communities
Raw Illumina MiSeq sequencing data was analyzed, using QIIME

| Comparison of functional bacterial genera response to short and long HRTs in control bioreactors and electro-bioreactors
To provide a clearer picture of the shifts we observed in the subpopulations of functionally relevant bacteria in our different reactors, two heat maps were constructed illustrating these differences (Figure 5a and b). To provide a more simplified comparison between the functional bacterial genera under different HRT conditions, we classified the HRT conditions into short HRT (6, 10 and 16 hr) as shown in Table 3 and long HRT (24, 50 and 75 hr) as shown in  Tolumonas. The fifth category contains bacterial genera that increase in abundance at Short but not long HRTs in electro-bioreactors compared to controls, and include Nitrospira, Bdellovibrio, and Candidatus Accumulibacter. The sixth category contains bacterial genera that increase in abundance at long but not short (more than at short

HRT) HRTs in electro-bioreactors compared to controls, and include
Pseudomonas, Streptococcus, Enterobacter, and Tolumonas. The seventh category contains bacterial genera that decrease in abundance at short but not long HRTs in electro-bioreactors compared to controls, and include Enterobacter and Tolumonas. The eighth category contains bacterial genera that decrease in abundance at long but not short HRTs in electro-bioreactors compared to controls, and include Nitrospira, Bdellovibrio, and Candidatus Accumulibacter.
At short HRT, there was a noticeable higher relative abundance of functional bacteria in reactors operated at short HRTs known to be associated with N-removal, such as Nitrospira and Janthinobacterium sp., P-removal, such as Dechloromonas sp. and sCOD reduction, such as Lactococcus, Flavobacterium, and Vogesella (Figure 6a and b). This indicates that growth and enrichment of functional bacterial species occurred in electro-bioreactors operated at short HRTs compared to the control (Table 3), resulting in better biological nutrient removal. This confirms that the metabolic activity of these species increased under the impact of electric field at current density 3 Am −2 and short HRT.
Other species which are functionally important such as Pseudomonas sp., a denitrifying species which has been previously confirmed to utilize different organic compounds and has been directly linked to sCOD removal in microbial fuel cells (Majumder et al., 2014), had higher observed OTU counts in electro-bioreactors operated at short HRT when compared to the control. Pseudomonas sp. has been reported to also be involved in bioremediation in municipal wastewater treatment (Wasi, Tabrez, & Ahmad, 2013). Additionally, an observed increase in OTU counts for microorganisms such as Hyphomicrobium sp. and Plesiomonas sp. in electro-bioreactors operated at short HRT than in control bioreactors (Table 3).
Hyphomicrobium is a denitrifying methylotroph, meaning that they reduce nitrate and use methane as a carbon and energy source.
Plesiomonas sp. is recently discovered to adsorb cadmium ions which can be applied in wastewater treatment plants in the future (Xue, Qi, Li, & Liu, 2016). The presence of an active population of fermenting bacteria that supply substrates to other functional groups is therefore T A B L E 3 OTU counts of various functional bacterial genera in control bioreactor (C) and electro-bioreactors (E) operated at short HRTs (6, 10, and 16 hr) and their corresponding role in nutrient and pollutant removal of fundamental importance for efficient N and P removal carried out by the activated sludge process in wastewater treatment systems.
Fermenting microorganisms use an internally balanced redox process in which the organic substrate becomes both oxidized and reduced.
Streptococcus, a glucose fermenting species, was found to be in slightly higher abundance in electro-bioreactors operated at short HRT compared to control bioreactors (Table 3). There was a slight increase in relative abundance of certain bacterial species in electro-bioreactors operated at short HRT which is originally present in low abundance as shown in Figure 6b, such as sulfur reducing bacteria (Thiobacillus), hydrogen oxidizing bacteria (Hydrogenophaga), recently discovered species to be associated with industrial production of polyhydroxybutyrate (Zobellella) (Ibrahim & Steinbüchel, 2010) and micropollutant removing bacteria such as aromatic hydrocarbons and BPA (Sphingobium) (Sasaki, Maki, Oshiman, Matsumura, & Tsuchido, 2005) (Table 3).
Interestingly, there was an observed increase in the relative abundance of a known beneficial bacteria Bdellovibrio sp. in electrobioreactors operated at short HRTs compared to control bioreactors (Table 3) In contrast to the genera mentioned above in which application of current enhanced their growth at short HRT's, relative abundance of the Enterobacter sp. decreased in electro-bioreactors at short HRTs. Enterobacter sp. are known as antibiotic resistant bacteria (Meyer, Saunders, & Blackall, 2006;Nielsen et al., 2010), and their presence or depletion is important in determining the efficiency of a wastewater treatment technology and effluent quality (Espigares, Bueno, Espigares, & Gálvez, 2006;Filipkowska, 2003). At long HRT, bacterial communities associated with N-removal such as Nitrospira sp. favored control bioreactors than electro-bioreactors as shown in Figure 6c and d, as their relative abundance was higher than in control bioreactors operated at HRT of 75 hr as shown in

| Reactor performance and physiochemical parameters under different HRTs
The highest differences in sCOD removal between the control bioreactor and the electro-bioreactor were at the ones operated under HRT of 6, 24, 50, and 75 hr. The decrease in sCOD removal efficiency in control bioreactors operated at short HRT could be related to the shorter contact time between the activated sludge and organic matter. In addition, the decrease in sCOD removal efficiency might also be due to the fact that some bacteria were washed out from the sequencing batch reactor when HRT was less than the facultative bacteria-generation time (Kapdan, 2005;Yang et al., 2006).
However, in electro-bioreactors operated at HRT of 6 hr, there was an increase in sCOD removal efficiency which is due to electrocoagulation process resulted from the impact of electric field (Giwa F I G U R E 6 Graph indicating differences in the relative abundance represented in OTU counts of functional bacterial genera between control bioreactors and electro-bioreactors: (a) high abundant bacterial genera at short HRTs, (b) low abundant bacterial genera at short HRTs, (c) high abundant bacterial genera at long HRTs, and (d) low abundant bacterial genera at long HRTs  Hosseinzadeh, Bidhendi, Torabian, Mehrdadi, & Pourabdullah, 2015).
The high removal of phosphorus in electro-bioreactors at all HRTs is not only attributed to biodegradation (via precipitation in the sludge) but also to the electro-deposition phenomenon through which phosphorus ions tend to deposit on the surface of the electrodes, mainly on the cathode, as previously reported (Giwa & Hasan, 2015;Ioan & Robescu, 2015;Klimiuk & Kulikowska, 2006).
In all reactors, it has been observed that there is unbalanced ammonium oxidation-to-nitrate and ammonium removal. These results agree with previous studies which assumed that the lack of balance between ammonium amount and nitrate formed in control bioreactors and electro-bioreactors could be due to partial nitrification, denitrification in aerobic conditions (Klimiuk & Kulikowska, 2006).
It has also been reported that NH 4 + -N removal in a single, aerated reactor is caused by complete autotrophic nitrogen removal over nitrite, known as the CANON process (Peng, Wu, Yu, Ai, & Fu, 2013).
The reported results here show that electro-bioreactors operated at low HRT of 6, 10, and 16 hr enhanced reactor performance in removing both PO 4 3− -P and NH 4 + -N, with very low measured concentrations in the effluent which did not exceed ≃0.01 and 0.05 mg L −1 , respectively. Taken together with results previously reported by our group (Zeyoudi et al., 2015), this confirms that optimizing current density and HRT-operating parameters in an electro-bioreactor enhances nutrient removal efficiency, resulting in cleaner effluent and high water quality.

| Functional bacteria in control and electrobioreactors in linkage to biological nutrient removal
HRT is considered as one of the most important operating parameters affecting the performance and microbial community of the bioreactor (González-Martínez et al., 2013;Ioan & Robescu, 2015;Wang et al., 2009Wang et al., , 2015Zhang et al., 2015). P-removal in electrobioreactors occurs due to electrocoagulation process (Giwa & Hasan, 2015) in addition to electric current stimulation effects on bacterial communities affiliated with P-removal. As shown in Figure 2b, high removal efficiency of PO 4 3− -P (97%-99%) was observed in all reactors operated at higher HRTs. Interestingly, higher HRT's were also associated with high OTU counts for Dechloromonas sp. (Figure 5) in all reactors. Furthermore, relative abundance of Dechloromonas were high even at low HRT's in electro-bioreactors compared to the control ( Figure 5) (Fujitani, Aoi, & Tsuneda, 2013). Recent findings by (Daims et al., 2015) reported that the genome of the Chemolithoautotrophic nitrifying bacterium encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. These findings point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities (Daims et al., 2015;Juretschko et al., 1998). Indeed, the only reactor with poor ammonia removal was the control reactor at HRT of 6 hr, and this reactor had the lowest Nitrospira counts (Figures 2c   and 5). All the other reactors containing higher relative abundance of Nitrospira sp. were associated with higher N-removal efficiency ( Figures 2c and 5).
sCOD indicates organic pollutants in the wastewater. Bacteria oxidizes organic compounds in the wastewater for their growth and metabolism (Henze, 2008). Therefore, the efficiency of wastewater treatment can be also assessed by measuring sCOD removal. As mentioned earlier, sCOD removal efficiency was high in all electrobioreactors compared to the control bioreactors ( Figure 2a). Here, sCOD removal efficiency in the control bioreactors ranged between 92% and 94% at all HRT tested, while sCOD removal reached 96% to 98% in electro-bioreactors at HRTs of 6, 24, 50, and 75 hr (Figure 2a).
Pseudomonas and Flavobacterium have been previously reported that they can remove sCOD (Abdel-Raouf, Al-Homaidan, & Ibraheem, 2012; Nasr, 2010). Additionally, there was an observed high removal of sCOD in electro-bioreactors operated at HRT of 6 hr which could be due to the electric current impact resulting in stimulating the growth of bacterial communities associated with sCOD reduction such as Lactococcus, Pseudomonas and Flavobacterium Nielsen, Nguyen, Meyer, & Nielsen, 2012;Park et al., 2007). Our results showed that there was a higher relative abundance of those genera in electro-bioreactors operated at HRT of 6 hr (5.8, 4.3 and 3.7%, respectively) than in control bioreactors (0.1, 1.1, and 2.1%, respectively). Indeed, species of the genus Lactococcus could produce lactate by fermentation of glucose. The ability of Lactococcus to degrade sCOD in trichloroethylene wastewater has been previously reported as well . Taken together, we propose that a dual mechanism of sCOD removal is occurring in the electro-bioreactor at the low HRT of 6 hr: An electrocoagulation process where organic compounds form coagulates that precipitate out of the wastewater in addition to biodegradation process where microorganisms stimulated by the current oxidize or sequester these organic compounds. This dual mechanism most likely holds true for all the other nutrients we measured in these experiments. Finally, this data represents the results that describe microbial communities that evolved when an initial sample of activated sludge is passed through a synthetic wastewater bioreactor under various operating parameters for 24 hr. The microbial communities that evolve if this was repeated with different starting material and real wastewater with different characteristics will most likely be different.

| CONCLUSION
Results showed that electro-bioreactors operated at CD of 3 Am −2 under different HRT conditions could effectively remove sCOD (96%-98%), PO 4 3− -P (96%-99%) and NH 4 + -N (99%) compared to control bioreactors at short HRTs. The relative abundances of functional bacterial genera varied depending on short and long HRT tested in both control bioreactors and electro-bioreactors. We also discussed changes observed in the microbial population structure and how they potentially relate to reactor performance and effluent quality. These are the first results to describe effects of varying HRT on microbial community structure in wastewater electro-bioreactors.

ACKNOWLEDGMENT
The authors thank Masdar Institute of Science and Technology for their financial support (Grant No. 13KAMA2). Also, authors are thankful to the team of Masdar City's MBR plant for supplying us with sludge samples that were used in this study.