Single-Stage Biolm-Based Total Nitrogen Removal in a Membrane Aerated Biolm Reactor: Impact of Aeration Mode, HRT and Scouring Intensity

High energy costs, organic carbon availability, and space limitation are some of the barriers faced by wastewater treatment processes. This research investigates the impact of membrane aeration mode, scouring intensity, and loading rate in a single-stage total nitrogen removal process in a membrane aerated biolm reactor (MABR). Under ammonia loading of 2.7 g N/m 2 .d, continuous process aeration led to 1.7 g NH 4 -N/m 2 .d and 0.8 g TN/m 2 .d removal, respectively. Conversely, intermittent (5/12 min on/off) aeration resulted in 35% less ammonia removal but 34% higher total nitrogen (TN) removal. The MABR under ammonia load of 1.6 g N/m 2 .d showed an enhanced euent quality with an average of 2.5 mg/L euent ammonia concentration. This nding highlights the nitrication potential of a ow-through MABR as a standalone treatment step without any downstream process. Also, slough-off, a common issue in the biolm process and was hypothesized to reduce the removal eciency, showed increased ammonia removal rates by 20%. The microbial analysis indicated the dominant AOB and NOB species as Nitrosomonas spp. and Nitrospira spp, respectively. Moreover, the relative abundance of denitrifying bacteria (40.5%) were found twice in intermittently-aerated MABR compared to the continuously-aerated one (20.5%). However, NOB and denitrifying bacteria relative abundances were comparable where continuous air was supplied.


Introduction
The extent of energy consumption in the biological wastewater treatment industry has become the center of attention in recent years. Population growth and the resulting higher contaminant loading is the motivation to establish energy-e cient approaches. In biological nitrogen removal processes, nitri cation and denitri cation pathways, the process's energy e ciency can be assessed through aeration in nitri cation and carbon demand in denitri cation. Total nitrogen removal using a conventional treatment requires substantial biological aeration (4.57 g O 2 /g NH 4 -N) and a carbon demand of 3.71 g organic carbon/g NO 3 -N (Tchobanoglous et al., 2014). Furthermore, processes such as carbon oxidation by aerobic heterotrophs and the use of low-e ciency blowers for oxygen supply in uence the energy balance negatively. Alternatively, membrane aerated bio lm reactor (MABR) can be considered to overcome the high energy demand associated with bubble aeration (Houweling et al., 2017b;Martin et al., 2012). MABR technology uses a gas transfer selective membrane to deliver oxygen to a bio lm attached to a membrane surface while other substrates like ammonia and carbon diffuse from the bulk liquid into the bio lm layers (Fig. 1).
Oxygen mass transfer rate (OTR) across the membrane is one of the most critical parameters characterized in MABRs. When the membrane is immersed in clean water with no bio lm grown on, gas ux through the membrane depends on the membrane mass transfer coe cient and the oxygen concentration gradient, in uenced by oxygen-water Henry's constant (Cote et al., 1989). However, in a wastewater treatment process where bio lm grows on the membranes' surface, bio lm plays a signi cant role in determining the oxygen concentration gradient. Bio lm oxygen demand, which is a function of bio lm characteristics and operational parameters such as other substrates concentration (nitrogen and carbon), in uences the oxygen gradient's extent within the membrane wall in a treatment process (Pellicer-Nàcher et al., 2013; Shanahan and Semmens, 2006). The OTR and OTE are calculated as per equations 1 and 2 (Côté et al., 2015): Where OTR is in g/m 2 .d, M O is oxygen molecular weight (32 g/mol), Q PF and Q PE are process gas feed and exhaust speci c ow rates (Nm 3 /m 2 .h), V m is the standard gas volume at standard temperature and pressure (STP) (0.0224 m 3 /mol), and X F and X E are molar fractions of oxygen in feed and exhaust gas.
MABR systems are becoming popular in biological carbon and nitrogen removal processes because of their capability of hosting a higher amount of biomass and, consequently, achieving higher removal rates while saving a considerable amount of oxygen (up to 70%) compared to conventional systems as well as smaller footprint (Houweling et al., 2017a). Several studies have shown the potential of MABR to address carbon removal, nitri cation (municipal and ammonium-rich industrial wastewater) (Da Silva Table 1 demonstrates, the few MABR studies implementing SND either have obtained low removal e ciencies or have applied longer HRTs (12-28 h) as well as higher C:N (5-18) ratios. However, the C:N ratios that were studied are much higher than the C:N ratio (< 3) that can be achieved in post-carbon redirection processes, hence further studies at lower C:N ratios are required to evaluate the feasibility of using chemically enhanced primary treated (CEPT) wastewater in denitri cation without adding any external carbon source. In addition, the longer HRTs considered in previous studies results in larger reactor volume; establishing a robust SND process at relatively lower HRTs (approximately 6-8 times lower than typical HRTs applied in the literature) is of great interest. Another challenge that is supposed to be addressed in this study is comparing MABR performance under intermittent versus continuous aeration mode. Although MABR has been proved as an energy saver in terms of aeration, no investigation has been conducted to examine mainstream SND e ciency in MABR under intermittent aeration condition except the few sidestream (ammonium-rich wastewater) nitritation studies (Ma et al., 2017; Pellicer-Nàcher et al., 2010).

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The counter-diffusional mass transfer pathway occurring within MABR bio lm layers is hypothesized to establish a robust SND system using CEPT wastewater at short HRTs. Overall, in this study, MABR's potential is explored to establish a simultaneous nitri cation-denitri cation system in the shape of a "single-stage bio lm-based total nitrogen removal system" low-strength municipal wastewater characterized by lower C:N ratio. The speci c objectives of this study include: 1. Implementing one-stage bio lm-based SND in MABR a) using low-carbon CEPT feed without adding any external carbon source and b) under quite short HRTs (2.5 h), 2. Identifying the maximum nitrogen removal e ciency using intermittent aeration and characterizing the trade-off between energy consumption and removal e ciency

MABR Pilot setup and operation
The study was conducted using four membrane-aerated bio lm reactor pilots with automatic control capability. Each pilot consisted of a 2L membrane tank and a junior Zeelung® membrane module (surface area of 0.14 m 2 ). Variable-speed peristaltic pumps (capacity range of 30-450 mL/min) were used to pump feed wastewater into the reactors. Compressed air was used as the process air source, and high-purity (> 99.998%) compressed nitrogen gas was used as the mixing and scouring gas throughout the study. The process air (15 mL/min) was supplied intermittently through the membranes, where it diffused into the bio lm and was used as an electron acceptor during the carbon and ammonia oxidation processes. The intermittent aeration sequence used was 5 min on and 12 min off operation. The mixing gas (170 mL/min) was directly supplied to the membrane tank continuously to replenish the media with a fresh substrate, whereas the scouring nitrogen gas (2000 mL/min) was supplied to the tank intermittently as according to the experimental design. Figure 2 represents a schematic view of the MABR pilot.
Two of the reactors, henceforth referred to as R1 and R2, were operated under the normal scouring condition and HRT of 2.5 h, while one reactor was supplied by intermittent aeration and one by continuous aeration. Normal scouring refers to performing scouring every four hours, with each scouring lasting for one minute. The second set of reactors, R3 and R4, were operated under no scouring condition and HRT of 4 h, one intermittently and one continuously aerated. Aeration, mixing, scouring, and feed-ow control sequences were programmed using Factory Talk® View software (Site Edition, CPR 9 SR 6). Each pilot was equipped with two correlated owmeters to measure gas ow rates and two pressure gauges to monitor inlet and outlet process air pressures.

Wastewater characteristics
The reactors were seeded with a nitrifying sludge obtained from an activated sludge plant (Vauxhall pollution control plant (PCP), London, Ontario, Canada). Sludge was screened using a 0.2 mm sieve and then seeded into the MABRs. The reactors were fed with CEPT wastewater from the Vauxhall PCP. 500 L of feed wastewater was delivered every week, and the feed was stored at room temperature over one week. The average feed ammonia concentration, sCOD concentration, and sCOD:N ratio were 16.9 ± 4.3 mg NH 4 -N/L, 33 ± 11 mg COD/L, and 2.2 ± 1.1, respectively.

Sample Analysis
Samples were collected twice a week from each MABR reactor and the feed wastewater. All samples were analyzed using Hach methods for NH 4 -N (method #10031), NO 3 -N (method #10020), NO 2 -N (method #10019), TCOD, and sCOD (method #8000). In uent TSS and e uent ML(V)SS were measured following standard methods (Rice et al., 2012). In addition, bulk liquid DO and pH (Orion 087003, Thermo Scienti c) were monitored twice per week. The off-gas oxygen fraction was monitored by connecting the oxygen analyzer device (MaxO 2 + AE, Maxtec) and the AMI oxygen analyzer (model 70R1) directly to the exhaust side of the membrane (Fig. 2). At the end of the study, bio lm samples were collected from each reactor to identify the relative abundance of microbial species on the bio lm (Molecular Research, MRDNA, Shallowater, TX, U.S.). Single-factor analysis of variance (ANOVA) was carried out in Excel to discern a signi cant difference between mean values, and a P-value < 0.05 is considered signi cant.

Batch Test
Two batch experiments were conducted to estimate the maximum nitri cation and denitri cation rates of a well-developed bio lm and compare the maximum total nitrogen removal vs. nitri cation rate under intermittent aeration vs. continuous aeration. The batch tests were conducted using CEPT e uent and the MABR pilot that had been operating for over eight months. Before a batch test was initiated, each MABR was drained entirely and loaded with 2 L of feed. For nitri cation batch tests, the feed stream was the CEPT wastewater similar to the one used in the long term study, whereas the CEPT wastewater was spiked with NaNO 2 and NaNO 3 to achieve 15-20 mg/L of nitrite and nitrate for the nitrite and nitrate denitri cation batch tests, respectively. All batch experiments were carried out at 22°C, and samples were collected every 30 minutes for NH 4 -N, NO 3 -N, NO 2 -N, sCOD, and DO analysis.

Results And Discussion
3.1 Impact of aeration mode on ammonia and total nitrogen removal: HRT of 2.5 h with membrane scouring Two MABRs were operated for 230 days, under the normally-scoured condition to minimize ammonia diffusion limitation to AOB. Scouring is a typical membrane operation strategy to keep bio lm thickness within an optimum range and minimize mass transfer resistance. Using scouring, ammonia diffusion within the bio lm layers will face less resistance, and consequently, higher ammonia removal e ciency is supposed to occur. In this work, the normal mode of scouring refers to 1 min long of scouring (2000 mL/min) with four times per day frequency. The ammonia loading rate in previous MABR mainstream wastewater studies ranged between 0.3-7 g/m 2 .d (Gilmore et al., 2013;Semmens, 2005). In this experiment, the ammonia loading rate was selected as 2.7 g/m 2 .d resulting in a short HRT of 2.5 h.
Although the loading rate of 2.7 is within the typical range for nitri cation systems, no studies have been found at a shorter HRT of 2.5 h. The shortest HRT used in the previous co-diffusional or counterdiffusional bio lm studies was 7.6 h, while the typical range was 12-28 h ( Table 1). Two different aeration modes, intermittent (5 min On/12 min Off) and continuous aeration, were applied to R1 and R2. reported relatively higher nitri cation rates of 1.2 and 2.6 g/m 2 .d in a hybrid MABR-activated sludge system where denitri cation occurred in the suspended sludge zone rather than the bio lm zone (Peeters et al., 2017).
One of the challenges reported regarding TN removal e ciency is outcompeting denitri ers by aerobic heterotrophs when C:N ratio of 5 and higher is applied (Iannacone et al., 2019). Using intermittent aeration mode in MABR, it is hypothesized that developing an environment for the denitri ers to outcompete the aerobic heterotrophic bacteria for the limited available carbon is a possible counterdiffusional pathway that would limit oxygen availability to the most exterior bio lm layer where the aerobic carbon oxidizing bacteria typically grows. Therefore, the signi cance of combining counterdiffusional bio lm and supplying intermittent aeration comes to play when dramatically decreased nitri cation and denitri cation rates can be seen in an MBBR (co-diffusional bio lm) under C:N ratio of 5.6, which caused aerobic heterotrophs overgrowth and consequently out-competing both nitri ers for oxygen and denitri ers for carbon (Iannacone et al., 2019). Table 2 summarizes the recent co-diffusional bio lm studies conducted to remove total nitrogen from municipal wastewater. In this study, the average oxygen transfer rate (OTR) and average bulk liquid DO was measured as 5.2 g/m 2 .d and 0.6 mg/L, respectively. The OTR values were 50% lower than the one observed in continuous aeration (11.7 g/m 2 .d); however, the bulk liquid DO was slightly lower, where the latter had an average DO of 0.8 mg/L. As a pathway indicator, the ratio of g O 2 consumed /g NH 4 -N removed was calculated as 4.7 and 6.9 g/m 2 .d in R1 and R2, respectively. In continuously-aerated MABR, the ratio of 6.9 is relatively higher than the stoichiometric oxygen demand for nitri cation (4.57 g O 2 consumed /g NH 4 removed ). As a result, although the amount of available carbon was relatively low, the carbon removal pathway included aerobic oxidation and denitri cation in continuously-aerated MABR.
It is also worth noting that, compared to intermittent aeration, a signi cant proportion (P<0.05) of the oxidized ammonia remained as nitrate in the continuously-aerated MABR (0.4 and 1 g/m 2 .d for R1 and R2, respectively) ( Figure 3a). Figures 3b and 3c also present ammonia and TN removal e ciency pro les over time. The results demonstrate that 27% more of the oxidized ammonia was removed as total nitrogen in R1 compared to R2. Remarkably, this correlation between ammonia removal and nitrate production explains the trade-off between intermittent vs. continuous operation: (i) with intermittent aeration: less ammonia removal and having nitrogen gas as the dominant end-product with a saving of 70% aeration intensity or (ii) with continuous aeration: higher ammonia removal rate with more nitrate as the end-product. This result has further strengthened the conviction that MABR can be used as an adaptable technology depending on the WWTP's treatment goal(s). In WWTPs, which consider ammonia removal as the primary target, and there is no restriction to meet e uent nitrate criteria, continuous aeration will be a wise choice since it can lead to higher ammonia removal e ciency. However, when total nitrogen removal is emphasized in a treatment plant, intermittent aeration can be a suitable strategy to achieve higher total nitrogen removal e ciency.
3.2 Impact of aeration mode on ammonia and total nitrogen removal: HRT of 4 h associated with no membrane scouring  from the reactor, and denitrifying activity reached almost zero (Fig. 4c). The collapsed denitri cation e ciency due to slough-off proved that the denitrifying population was developed on the bio lm's outer layers. In addition, the fact that the slough-off occurred in the continuously-aerated reactor con rms the overgrowth of heterotrophs and the signi cance of scouring to control the bio lm thickness. Under the loading of 1.6 g/m 2 .d, continuously-aerated non-scoured MABR (R4) achieved ammonia removal rate of 1.4 g/m 2 .d (86%), which resulted in an average e uent ammonia concentration of 2.5 mg/L that is aligned with discharge limits. While continuously-aerated non-scoured MABR showed the full capability of removing ammonia with no need for downstream processes, intermittently-aerated MABR (R3) achieved 0.9 g/m 2 .d (56%) ammonia removal rate. In terms of total nitrogen removal, R3 showed a higher TN removal capability of 70% with 30% nitrate accumulation, while a much higher nitrate accumulation (87%) was observed in R4. It should be noted that all the R4 data discussed so far include the whole experiment containing before and after the slough-off phenomenon.
Regarding the performance of R4, ammonia removal e ciency can be divided into two different periods, before and after the slough-off phenomenon. Before slough-off occurred, the ammonia removal e ciency was 75% in R4, whereas 90% e ciency was observed after the slough-off. Regarding denitrifying activity, TN removal e ciency dropped from 65-14%, and consequently, residual nitrate increased from 35-85%. These ndings indicated that although denitrifying activity was destroyed due to slough-off, a 20% increase in ammonia removal e ciency was seen, which might be the result of decreased diffusion resistance for ammonia into a thinner bio lm after the slough-off. Slough-off is a common issue in any bio lm process. It is typically accompanied by loss of nitri cation activity; however, this study's nding showed that it does not reduce nitri cation activity and enhanced ammonia oxidation rate. Increased nitri cation rate after the slough-off can con rm the reliability of MABR in terms of ammonia removal.
As discussed earlier, although extensive research has been carried out on SND in conventional bio lm systems, very few numbers of MABR studies explored the possibility of implementing a bio lm-based SND process, particularly for carbon-captured municipal wastewater. Among such MABR studies, some used hybrid systems (which do not support a bio lm-based process), and others have been restricted to apply very long HRTs, higher in uent ammonia concentration, and higher C:N ratios (Table 1). For example, Lin et al. (2016) conducted a study that examined the potential of MABR in establishing SND. They reported a 62% TN removal e ciency at C:N of 3 while HRT was 24 h. However, the same TN removal e ciency was achieved in this study by the HRT of 2.5 h, which translates into a faster process by almost ten times. Hence, we hope that this study's results could eliminate the research gap in using MABR to establish a robust one-stage TN removal system. Table 3 and Fig. 5 summarized the whole performance of the four MABRs operated under different aeration modes, scouring modes, and HRTs. Comparing ammonia and total nitrogen removal rates and e ciencies in two continuously-aerated MABRs (R2 and R4) but under normal and non-scoured conditions showed a 20% more ammonia removal in R2 compared to R4. This agrees with the nding obtained in (Mehrabi et al., 2020), where normal scouring led to a higher ammonia removal e ciency than the non-scoured MABRs. The higher ammonia removal corresponding with normal scouring is expected to relate with the scouring of the outer heterotrophic carbon oxidizing bio lm layer resulting in a reduced mass transfer resistance for ammonia diffusion through bio lm layers. Another interesting point that came off this experiment is scouring versus non-scouring in TN removal e ciency of the continuously-aerated MABRs. Under continuous aeration condition, the TN removal e ciency remained comparable despite the difference in scouring modes. This observation is quite different from the results reported in the intermittently-aerated reactors (R1 and R3) and also reported by Mehrabi et al. (2020) that indicated a higher TN removal rate of non-scoured MABRs compared to normally-scoured MABRs when intermittent aeration was supplied. That might be justi ed as when continuous air is introduced to the process, the scouring has no impact on total nitrogen removal e ciency due to the excess air available within the bio lm layers. This unlimited oxygen availability challenges developing an extensive anoxic bio lm layer on the MABR. Typically, the oxygen transfer rate is controlled by the substrate (ammonia and carbon) availability and biomass demand. However, is the observation in this study indicates that the nature of aeration supply mode (intermittent versus continuous) through the membrane also dictates the biochemical environment. Continuous aeration encourages the nitrifying organisms and limits the anoxic zone's availability as a desirable environment for denitrifying activity; however, further bio lm micro-level pro ling is required to con rm this observation.

Determination of maximum speci c substrate removal rate
Batch experiments were carried out to characterize the performance of the MABRs to remove ammonia and total nitrogen and identify the maximum removal potential of the system. The ammonia oxidation and denitri cation rate studies were carried out using CEPT wastewater and spiked CEPT wastewater, respectively (as described in Sect. 2.3). Figures 6a and b present the ammonia pro le data obtained during the nitri cation batch tests. The batch test showed ammonia removal rates of 3.5 and 5.4 g/m 2 .d in R1 and R2 (intermittent and continuously-aerated reactors at HRT of 2.5 h), respectively. As can be seen, the maximum potential of both MABRs is 3.2 times higher than the rates observed in the long-term experiment, representing the substantial nitri cation capability of the MABRs if they were run at higher loading rates in a batch mode using a similar HRT of 2.5 h. In non-scoured MABRs (Fig. 6b) where the bio lm was developed on a long-term ammonia loading of 1.6 g/m 2 .d, ammonia removal rate obtained in batch tests were 2.3 and 2.9 g/m 2 .d in intermittently and continuously-aerated MABRs, respectively.
Again, the maximum capability of the non-scoured MABRs is higher than the long-term results by 2-2.5 times; however, it is relatively lower than the maximum capability of the normally-scoured MABRs.
Denitri cation rates were also identi ed in R1 and R2. Both nitrite and nitrate removal rates have been identi ed as the denitri cation capacity of the system. The nitrite removal rate (Fig. 6c) was measured as 5 and 3.5 g/m 2 .d under intermittent and continuous aeration. However, the corresponding nitrate removal rates were lower (2 and 1.8 g/m 2 .d, respectively) (Fig. 6d). The exciting nding is the substantially higher potential (two times) of the system to remove nitrite compared to the nitrate in both reactors. This observation might be due to the concept of bio lm-based SND, which grows both nitri ers and denitri ers on the same medium. When denitri ers grow adjacent to the nitri ers, they might have the better capability to compete for the NOB for nitrite compared to the systems that denitri ers mostly grow in suspended activated sludge. As a result, in long-term treatment, the denitrifying population might selectively improve its nitrite reduction capability rather than nitrate. However, due to the high amount of available air in R2, the denitri cation rate (including both nitrite and nitrate) is relatively lower than R1, which shows the potential of intermittent aeration in MABR to enhance denitri cation activity via nitrite over NOB.

Impact of aeration on the microbial community of bio lm
To further characterize the biological process, the microbial population grown on the surface of membrane bres were analyzed. The bacterial population are presented in the phylum, class, and specieslevel in Fig. 7. At the phylum level (Fig. 7a), the dominant phylum, which accounted for more than 50% of the population, is Proteobacteria, including Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria classes, which was reported as the dominant phylum in many WWTPs involved with biological nitrogen and carbon removal processes. Betaproteobacteria and Gammaproteobacteria classes were identi ed as related bacteria to nitrogen and organic matter removal. Besides, the AOB genera of Nitrosomonas belongs to Betaproteobacteria, which is the dominant AOB genera in both R1 and R2. Bacteroidetes and Chloro exi, which were identi ed as two of the most popular phyla in both reactors, were observed as subdominant phyla in several WWTPs (Cydzik-Kwiatkowska and Zielińska, 2016; Gong et al., 2020;Tchobanoglous et al., 2014). The aerobic phylum of Nitrospirae grew in a signi cantly higher amount under continuous aeration rather than intermittent, which is consistent with the species result reporting Nitrospira sp as the most dominant species in R2 (Fig. 7c). Since unlimited oxygen was supplied to the reaction zone in continuous aeration, it justi es the higher ammonia removal e ciency and higher residual nitrate in R2. Therefore, at the species level, respective AOB and NOB relative abundance (RA) were 10.3% and 20.7% under continuous aeration (R2); however, the relative abundance of AOB and NOB were 1.3% and 1.8% under intermittent aeration (R1). This nding highlights the fact intermittent aeration signi cantly limits nitri ers' growth, which might have simultaneous positive and negative impacts on the process e ciency, as previously discussed. The lower NOB relative abundance in intermittently-aerated MABR was in agreement with previous works that reported a decreased nitrite-oxidizing bacterial (NOB) activity and growth rate under intermittent aeration. The authors reported Nitrospira as the dominant NOB species rather than the Nitrobacter that is typical for continuous aeration systems (Mota et al., 2005;Yang and Yang, 2011). Similarly, another study indicated that in the bio lm regions with oxygen scarcity, Nitrospira was found as the dominant species, which has a lower substrate utilization rate than Nitrobacter (Downing and Nerenberg, 2008).
The other striking result of the microbial analysis data is the signi cantly different relative abundance of nitri ers vs. denitri ers in intermittently-aerated MABR vs. the continuously-aerated one. The relative abundance of denitrifying bacteria was measured as 40.5% in R1, which is two times higher than R2 (20.5%). It is worth mentioning that 16% and 6% of denitrifying bacteria comprised of sulphur-oxidizing bacteria such as Sulfurisoma sediminicola in R1 and R2, respectively, can use nitrate as an electron acceptor and convert it to nitrogen gas. In general, R2 hosted a narrower range of microbial populations, mostly aerobic microorganisms, while R1 grew a vast range of different species, mostly anoxic/anaerobic with denitrifying capability as Sulfurisoma sediminicola, Trichococcus pasteurii, Thiothrix disciformis, and Methyloversatilis. The higher percentage of denitri ers and higher total nitrogen removal e ciency observed in R1 con rms the hypothesis of a well-developed anoxic region within the intermittentlyaerated bio lm. In other words, denitri ers could outcompete the NOB population for nitrite due to the favourable anoxic condition; however, NOB and denitri ers relative abundances were almost equal in R2.
Besides, while none of the reactors were seeded with anammox bacteria, it was interesting to nd 0.67% and 0.9% of anammox bacteria in R1 and R2, respectively. Figure 8 shows the relative abundance of different functional populations in each MABR.

Impact of bio lm sample extraction method
For microbial analysis, three bio lm extraction methods from the membrane were utilized. The methods include Qiagen Powersoil DNA extraction kit, Ultrasonic bath, and Vortex Mixer were examined. In both ultrasonic bath (30 min, 70 kHz) and vortex mixer (3 min, 2500 rpm), distilled water was used as the solvent. According to the analysis results, signi cant differences were reported in the relative abundance of microbial populations using three different extraction methods. The relative abundance of AOB, NOB, and denitrifying bacteria for ultrasonic bath and vortex mixer methods was detected as approximately 50% lower than using the Qiagen Powersoil DNA extraction kit RA of denitrifying bacteria using vortex mixer in R1 (Fig. 9). Regardless of the negative or positive error percentage, both ultrasonic bath and vortex mixers were found highly unreliable methods to extract the bio lm sample from the media comparing to the Qiagen Powersoil DNA extraction kit.

Conclusion
In this study, a single-stage bio lm-based total nitrogen removal was established to treat CEPT wastewater using MABR at short HRT of up to 2.5 h. The obtained results underlined the importance of aeration mode and HRT on different nitrogen compounds removal and accumulation rates through the bio lm. Continuous aeration led to higher ammonia removal as well as accumulating nitrate as the endproduct. On the other hand, although intermittent aeration resulted in less ammonia removal, TN removal was identi ed as the dominant pathway under this aeration mode, which showed the capability of establishing a single-stage bio lm-based total nitrogen removal process. Based on the ammonia loading vs. removal rate observed in the MABR pilot, ammonia removal achieved the discharge limit (2.5 mg/L), highlighting the possibility of operating ow-through MABR as the only treatment step without any downstream process. Another highlighted nding was a 20% increase in ammonia removal rate after the slough-off occurred in R4, most probably, due to the higher ammonia ux into the bio lm resulting from reduced diffusion resistance for ammonia molecules that can address the concerns regarding the common slough-off issue in MABR.
Taken together, the results in this study stand for the fact that the unique counter-diffusional bio lm formed in MABR facilitates one-stage bio lm-based total nitrogen removal by hiring different strategies such as aeration mode and HRT. Therefore, MABR would enable different wastewater treatment plants to ful ll the treatment targets. In plants that aim to remove only ammonia, a combination of non-scoured conditions and continuous aeration might be a proper alternative to achieve higher ammonia removal e ciency while no energy is consumed to scour the bio lm. This strategy might have a high risk of slough-off, which can be considered a bene t since it increases the removal e ciency when the main target is ammonia removal. However, for the WWTPs requiring total nitrogen removal, intermittent aeration should be considered as one of the necessary design parameters.

Declarations
Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.    The relative abundance of the different microbial population in R1 and R2 The relative abundance of the functional microbial population using different bio lm extraction methods in R1 and R2