Coupling membrane electro-bioreactor with anammox process to treat wastewater at low temperatures

2.1 Influent (feed wastewater) characteristics

Synthetic wastewater with similar characteristics was used to feed all operated reactors. The compositions of synthetic wastewater were previously reported at Lagum (Lagum, 2021). Table1 demonstrates the synthetic influent’s characteristics.

2.2 Experimental set-up

The MEBR-anammox (Fig. 1d) assembly composed of a PVC vessel and two cylindrical electrodes (a stainless steel cathode and an aluminum anode separated by a distance of 5 cm) placed around a PVDF hollow fiber (HF) ultrafiltration membrane module (GE, Zenon Membrane Solution, Canada) (Lagum and Elektorowicz, 2022). The MEBR-anammox had dimensions of 14 cm × 15 cm × 20 cm, a total reactor volume of 12.5L, and a hydraulic working volume of 7 L. Hydraulic retention time (HRT) and sludge retention time (SRT) of 24 h and 20 d were preserved in the reactor, respectively (Table 2). The MEBR-anammox was fed synthetic wastewater, whose characteristics were reported in Table 1, at a constant flow rate of 7 L/d via a peristaltic pump (Easy-load® Model 77202–55, Cole-Parmer Instrument, USA). The concentration of dissolved oxygen (DO) fluctuated between 0.1 and 1 mg/L due to air supply and electric current operation (Ibeid and Elektorowicz, 2021).

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Fig. 1

Schematic diagram of continuous-flow reactors: (a) conventional MBR (control), (b) MEBR (Stage I), (c) MEBR-SND (Stage II), and (d) MEBR-anammox (Stage III).

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The electrodes were perforated, where the anode had a porosity and an effective area of 40% and 520 cm², respectively. The membrane, characterized by a pore size of 0.04 μm and an effective filtration area of 0.047 cm², was vertically positioned in the middle of the reactor. Two air-stone diffusers were fixed at the bottom of the bioreactor under the membrane unit and around the electrodes, in order to supply the oxygen required for aerobic nitrifiers and to ensure sufficient mixing of the sludge liquor. The electrodes were attached to a digital DC-regulated power supply (Kepco BOP 50-2D Model, 0–30 V, 0–3 A, South Korea) and an electronic time switch (Model 5500, Control Company, USA). The electronic switch had the potential to deliver an optimal and constant CD of 14.85 A/m² with a periodic operational regime of 5 min ON and 10 min OFF (Bani-Melhem and Elektorowicz, 2011). This CD level maintained a pH of 7–8 and preserved high microbial activity (Lagum, 2022). Maintaining adequate DO and redox levels lets the MEBR-anammox system fluctuate between aerobic, anoxic, and anaerobic conditions to generate proper environments for all diverse types of N-removing bacteria (Adam and Elektorowicz, 2018). In MEBR-anammox, nitrifiers transform $\mathrm{N}{\mathrm{H}}_4^{+}$ to $\mathrm{N}{\mathrm{O}}_2^{-}$ and then the anammox consortium use the produced $\mathrm{N}{\mathrm{O}}_2^{-}$ to convert $\mathrm{N}{\mathrm{H}}_4^{+}$ directly to diatomic nitrogen (N₂) gases. Denitrifying bacteria transform the remaining $\mathrm{N}{\mathrm{O}}_3^{-}$ (formed as metabolic end-products of the anammox process) to N₂ gas.

2.3 Operating conditions

MEBR operation was divided into three distinct stages (Table 2), where the results of each stage were compared with those of a conventional MBR, which served as a control test. Stage I was conducted using a nitrifying MEBR and a nitrifying MBR. Both reactors were run simultaneously for one SRT cycle without DC induction, to demonstrate their similar characteristics. Then, two cylindrical perforated electrodes were introduced to the MEBR (Fig. 1b) to assess the performance of the newly designed system. Both MEBR and MBR operated with the same membrane modules whose characteristics were reported in Lagum (Lagum, 2019), influent flow rates of 7L/d, as well as HRTs and SRTs of 24 h and 20 days, respectively. The reactors at this stage were run for 2 months.

In Stage II, both MEBR (Fig. 1c) and MBR were operated with the same membrane modules, flow rates, HRTs and SRTs as in Stage I. Nevertheless, air and CD supplies make MEBR distinctive in design, where its design could generate simultaneous nitrification and denitrification conditions (Table 2). The concentration of DO was controlled via two pivotal factors: air supply and CD applied to the MEBR. The electrical circuit's contact regime (i.e., alternating ON/OFF) with an electronic timer regulates the oxygen reduction potential (ORP) and DO values. To attain this, the airflow and electrical current supply were monitored through flowmeters (Gilmont® Model GF-2500) and the voltage regulator of the DC power unit, respectively. In addition, the temperature was decreased (i.e., 15 °C) to examine the performance of the new MEBR design at these temperature conditions. Both reactors were operated in continuous-flow mode at this temperature for 2 months.

In Stage III, the MEBR was retrofitted to accomplish anammox reactions, attaining a MEBR/anammox system. The MEBR/anammox design was related to the MEBR system (Stages I and II) in terms of feeding composition, anode and cathode materials, electrode spacing, membrane module, as well as applied HRT and SRT. Nonetheless, the concentration of mixed liquor suspended solids (MLSS), CD level, exposure mode of electricity, the number of air diffusers, and the level of aeration were modified in order to adapt the anammox biomass. The anammox sludge was taken from a sidestream anammox enrichment unit (Adam and Elektorowicz, 2018), and added to the sludge of MEBR at the ratio of 1:2. Only two air distributors were kept to facilitate the growth of autotrophic nitrifiers without disturbing anammox and denitrifying bacteria .

The implementation of a low DO and adequate CD permits the development of simultaneous nitrification, anammox, and denitrification (SNAD) processes in the same vessel. The CD level at this stage increased due to the addition of anammox biomass and voltage adjustment. This increase in the CD was done to supply sufficient electrons to prompt bacterial metabolism and cell growth at low temperatures. Elektorowicz (Elektorowicz et al., 2016) stated that the CD magnitude has a direct correlation with MLSS or the concentration of sludge in the bioreactor. The temperature was additionally reduced in this stage to investigate the performance of the novel MEBR-anammox design at low-temperature conditions. Both MEBR-anammox and its comparative MBR were run for 2 months at a temperature of 10 °C.

2.4 Analytical methods and measurements

The concentrations of COD, orthophosphate ( ${\mathrm{P}\mathrm{O}}_4-\mathrm{P}$ ), ammonia nitrogen ( $\mathrm{N}{\mathrm{H}}_3^{+}-\mathrm{N})$ , nitrite-nitrogen ( $\mathrm{N}{\mathrm{O}}_2^{-}-\mathrm{N}$ ), nitrate-nitrogen ( $\mathrm{N}{\mathrm{O}}_3^{-}-\mathrm{N}$ ), TN and alkalinity were measured via spectrophotometry (Hach DR2800). These measurements were performed according to the instructions of the manufacturer by the Hach 8000, 10209, 10205, 10206, 10207, 10208, and 10,239 methods, respectively. DO, ORP and electrical conductivity (EC) were evaluated via HQ40d Multi-Parameter Meter (Hach, USA) with multiple IntelliCAL™ electrodes. Hydrogen ions (pH) and temperature were measured with a pH Probe (Fisher Scientific Accumet Basic AB15 pH Meter, USA). Total suspended solids (TSS) and volatile suspended solids (VSS) were determined by the gravimetric method in accordance with Standard Procedures (2540 D and 2540 E, respectively (APHA 2012).

3.1 Impact of temperature on the performance of MEBR and MBR – Nutrient removal

3.1.1

3.1.1 Carbon removal

Stage I: Operational Temperature (20 ± 2 °C)

Fig. 2a exhibits the COD concentration profiles of the influents and effluents of MEBR and MBR over the experimental period at room temperature (20 ± 2 °C). Both reactors reached substantial COD removal efficiencies at ambient temperature (99.43% for MEBR and 95.55% for MBR, respectively). The final effluent concentrations in both tested MEBR and MBR were as low as 1.60 mg COD/L and 12.45 mg COD/L, respectively. The lower COD concentration in the MEBR effluent demonstrates the beneficial influence of the EK processes on the removal of refractory organics.

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Fig. 2

Influent and effluents COD concentrations of different configurations of MEBRs and conventional MBR at various temperatures: (a) temperature = 20 ± 2 °C (Stage I), (b) temperature = 15 ± 2 °C (Stage II), and (c) temperature = 10 ± 2 °C (Stage III). The results were reported after the equilibrium day.

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Stage II: Operational Temperature (15 ± 2 °C)

Fig. 2b shows the COD concentration profiles of the influents and effluents of MEBR and MBR over the experimental period at temperature (15 ± 2 °C). It could be observed that the reduction of temperature from 20 °C to 15 °C did not significantly influence the COD removal in both treatment reactors. There was only a noticeable reduction in the removal of COD in the first two weeks of operation related to the biomass, which has not been adapted to this temperature change. However, after 3 weeks of operation, a significant COD removal of 78.89% and 86.04% were detected in the MBR and MEBR, respectively. At the end of the operational period, the removal of COD in both MBR and MEBR reached up to 87.15% and 91.80%, respectively. The concentrations of COD at a temperature of 15 °C in the treated effluents were 35.97 and 22.95 mg/L in the MBR and MEBR, respectively.

Stage III: Operational Temperature (10 ± 2 °C)

The variations of COD in the influents and effluents of MEBR-anammox and MBR over the experimental period at a temperature of 10 °C are presented in Fig. 2c. The results showed that the temperature decrease from 15 °C to 10 °C did not dramatically disturb the carbon removal process in the MEBR-anammox. In contrast, the MBR temperature decrease led to 50% COD removal efficiency. On some operational days, the MBR experienced COD removal absence due to foaming. MEBR-anammox achieved almost 90% COD removal efficiency at the end of the operation period (Day 59). The effluent concentrations of COD in both MBR and MEBR-anammox were 122.17 and 24.95 mg/L, respectively.

3.1.2

3.1.2 Phosphorus removal

Stage I: Operational Temperature (20 ± 2 °C)

Fig. 3a presents the variation of phosphorus (as orthophosphate, ${\mathrm{P}\mathrm{O}}_4-\mathrm{P}$ ) in the influent, MEBR and MBR effluents over operation time at room temperature (20 ± 2 °C). The P influent concentrations fluctuated between 16 and 20 mg ${\mathrm{P}\mathrm{O}}_4-\mathrm{P}$ /L. The lowest P concentrations in MEBR and MBR effluents at ambient temperature were 0.7 and 7.49 mg/L, respectively. The corresponding P removal efficiencies in both MEBR and MBR were therefore 96.26% and 59.51%, respectively. It was noted that after a few days of MEBR operation, P removal was already above 80%, whereas the maximum removal efficiency of the MBR in the whole operation period did not exceed 60%. Nevertheless, this removal percentage in the MBR was only attained as a result of the addition of aluminum trivalent cations (i.e., aluminum sulfate, Al₂ (SO₄)₃·18H₂O) to support P removal. Such significant P removal by MEBR is ascribed to the simultaneous biological (i.e., polyphosphate-accumulating organisms (PAOs) proliferation) and electrochemical processes taking place in the reactor (Lagum, 2021).

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Fig. 3

Influent and effluents P concentrations of different configurations of MEBRs and conventional MBR at various temperatures: (a) temperature = 20 ± 2 °C (Stage I), (b) temperature = 15 ± 2 °C (Stage II), and (c) temperature = 10 ± 2 °C (Stage III). The results were reported after the equilibrium day.

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Stage II: Operational Temperature (15 ± 2 °C)

Fig. 3b demonstrates P concentration in the influents and effluents of MEBR and MBR over the operation period at temperature (15 ± 2 °C). It could be observed that the reduction of temperature from 20 °C to 13 °C did not significantly disturb P removal in the MEBR system where ≥ 90% efficiency was obtained. P removal by MEBR at this stage (Stage II) showed similar trends to Stage I. Nevertheless, the MBR removal efficiency, even with the addition of coagulant agents, was severely influenced. The MBR removal did not exceed 30% efficiency. These findings suggest that the conventional MBR might be susceptible to temperature changes, resulting in minimal P removal ability.

Stage III: Operational Temperature (10 ± 2 °C)

Fig. 3c shows P variations in the influent and effluents of MBR and MEBR-anammox over the operational period at 10 °C. The integration of anammox sludge into MEBR resulted in low P removal efficiency in the first two weeks. Subsequently, the CD was increased from 12 to 15 A/m² by increasing the voltage gradient, where the MEBR-anammox demonstrated enhancement in the removal of orthophosphate. The lower P concentration in the effluent of the MEBR-anammox system points to the positive synergistic effects of electrokinetic and biological processes on P removal. The concentrations of orthophosphate in the treated effluents of MBR and MEBR-anammox at the end of the operational period were 15.95 and 0.8 mg/L, respectively (Fig. 3c). The corresponding removal efficiency of the MBR and MEBR-anammox reached 12.36% and 95.60%, respectively.

3.1.3

3.1.3 Nitrogen removal

Stage I: Operational Temperature (20 ± 2 °C)

Ammonium ( $N{H}_4^{+}$ ) and total nitrogen (TN) removal

Fig. 4a shows the variation of $\mathrm{N}{\mathrm{H}}_4^{+}$ profiles in the influent and effluent of MEBR and MBR over the operation period at room temperature (20 ± 2 °C). This stage established that the MBR at ambient temperature could remove $\mathrm{N}{\mathrm{H}}_4^{+}$ compounds to about 85.89%. Nevertheless, the MEBR enhanced $\mathrm{N}{\mathrm{H}}_4^{+}$ removal to almost 100% efficiency. The lowest concentrations of $\mathrm{N}{\mathrm{H}}_4^{+}$ in the MEBR and MBR effluents were 0.36 and 2.95 mg/L, respectively (Fig. 4a). Furthermore, after 10 days of MEBR operation, $\mathrm{N}{\mathrm{H}}_4^{+}$ removal was above 70%, while the MBR took some time to reach stable condition and sufficient $\mathrm{N}{\mathrm{H}}_4^{+}$ removal. Such superiority in the $\mathrm{N}{\mathrm{H}}_4^{+}$ removal efficiency and nitrification performance by the MEBR is attributed to the enrichment of a vigorous nitrifying community within the process (Lagum and Elektorowicz, 2022).

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Fig. 4

Influent and effluents ${\mathrm{NH}}_4^{+}$ , ${\mathrm{NO}}_3^{-}$ and TN concentrations of different configurations of MEBRs and conventional MBR at various temperatures: (a, b) temperature = 20 ± 2 °C (Stage I), (c, d) temperature = 15 ± 2 °C (Stage II), and (e, f) temperature = 10 ± 2 °C (Stage III). The results were reported after the equilibrium day.

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In this stage, TN removals were found to be 76.45% and 77.66% in the MBR and MEBR, respectively. The TN concentrations in the effluents were found to be 18.69 mg/L and 14.85 mg/L in the MBR and MEBR, respectively (Fig. 4b). This indicates that high DO concentrations (5–6 mg/L for MBR and 3.2–4 mg/L for MEBR, respectively) did not permit to eliminate nitrate, which could be accountable for the moderate TN removal efficiencies. Average concentrations of nitrates (as ${\mathrm{NO}}_3^{-}-\mathrm{N}$ ) in the treated effluents were 15.8 and 12.1 mg/L in the MEBR and MBR , respectively.

Stage II: Operational Temperature (15 ± 2 °C)

In Stage II, N removal was evaluated based on two tests: $\mathrm{N}{\mathrm{H}}_4^{+}-\mathrm{N}$ and $\mathrm{N}{\mathrm{O}}_3^{-}-\mathrm{N}$ . The measurements of $\mathrm{N}{\mathrm{H}}_4^{+}-\mathrm{N}$ and $\mathrm{N}{\mathrm{O}}_3^{-}-\mathrm{N}$ were performed on a daily basis and consistent results were reported as follows:

Ammioinum ( $N{H}_4^{+})$

Temperature reduction from 20 °C to 13 °C did not significantly affect the nitrification process in the MEBR (i.e., 10% decrease), while the MBR experienced almost complete deactivation of the biological nitrification process. The marginal decrease of $\mathrm{N}{\mathrm{H}}_4^{+}$ removal in the MEBR was compensated by average denitrification of 93.50%, demonstrating the efficiency of simultaneous nitrification and denitrification (SND) processes. The concentrations of $\mathrm{N}{\mathrm{H}}_4^{+}$ in the treated effluents at a temperature of 15 °C were 2.8 and 19.95 mg/L in the MEBR and MBR, respectively (Fig. 4c). The corresponding $\mathrm{N}{\mathrm{H}}_4^{+}$ removal efficiencies of the control MBR and MEBR were therefore 33.50% and 90.67%, respectively.

Nitrate ( $N{O}_3^{-})$

The $\mathrm{N}{\mathrm{O}}_3^{-}$ concentration profiles of the influent, as well as in the effluents from MEBR and MBR over the whole operational period at temperature (15 ± 2 °C) are shown in Fig. 4d. The adjustment of DO and electrical current application (Table 2) in the MEBR enhanced the performance of the nitrification efficiency at a temperature of 15 °C and permitted, after it, the denitrification to happen. Nevertheless, low temperature affects both nitrification and denitrification processes in the MBR. The average $\mathrm{N}{\mathrm{O}}_3^{-}$ concentration in the treated effluent from the MEBR reactor was 1.53 mg/L, with the lowest $\mathrm{N}{\mathrm{O}}_3^{-}$ concentration of 0.18 mg/L. Contrariwise, the average $\mathrm{N}{\mathrm{O}}_3^{-}$ concentration in the effluent from the MBR was 6.18 mg/L, with an $\mathrm{N}{\mathrm{H}}_4^{+}$ accumulation in the effluent as a consequence of the suppression of the biological nitrification activity and reactor equilibrium at 15 °C. It may be noted that the influence of temperature on the nitrification process was more evident than in the denitrification process.

Stage III: Operational Temperature (10 ± 2 °C)

Ammonium ( $N{H}_4^{+})$ and total nitrogen (TN) removal

Fig. 4e indicates the $\mathrm{N}{\mathrm{H}}_4^{+}$ profiles in the influents and effluents of the MEBR-anammox and the MBR over the whole operational period at a low temperature of 10 °C. The acclimatization of anammox biomass to the MEBR system revealed almost complete $\mathrm{N}{\mathrm{H}}_4^{+}$ removal at low temperatures. Nevertheless, the removal efficiency of $\mathrm{N}{\mathrm{H}}_4^{+}$ by the MBR was significantly affected, resulting in $\mathrm{N}{\mathrm{H}}_4^{+}$ effluent concentration of 26.95 mg\L at low temperatures (Fig. 4e). Due to the ability of the MEBR-anammox to support the growth of different N-removing bacteria, the effluent TN concentration and removal efficiency were 7.45 mg\L and 88.91%, respectively. By contrast, the MBR demonstrated high TN concentrations (46.38 mg\L) in the effluent (Fig. 4f).

3.2 Comprehensive discussion – Nutrient removal at various temperatures

Carbon removal

Carbon (C) removal through biomass oxidation was very high in MEBR and MBR at ambient temperatures. The low COD concentrations in the effluent recognize the excellent carbon degradation capabilities of both reactors at ambient temperatures (12.45 mg/L and 1.6 mg/L for the control MBR and MEBR system; respectively, Fig. 2a). Furthermore, both reactors demonstrated adequate COD removal efficiency even from the process start-up (≥80%). These results are consistent with the findings reported in previous studies (Adam and Elektorowicz 2017; Hasan et al., 2014; Wei et al., 2012). The advanced removal efficiency of COD (effluent concentration ≤ 2 mg/L) by the MEBR system was owing to the high performance of microbial flocs, which was in agreement with the results obtained for biomass activities reported by (Adam and Elektorowicz (2017)). It could be explained that the comprehensive removal of organic matter in the MEBR system is a result of the synergistic effects between biodegradation, biosorption, adsorption by electrostatic attraction, electrooxidation and electrocoagulation processes.

In Stage II, the removal efficiency of COD at a temperature of 15 °C showed almost the same removal trends as in Stage I (i.e., 20 °C). COD removal ranging between 85% and 87% was observed in the MBR. The MEBR, however, achieved the highest removal efficiency (≥92.3%) at the same operational temperature. The MEBR at this stage (Stage II) operated at low DO concentrations (0.5–2.5 mg\L) where denitrifier communities were encouraged at this DO level, contributing to additional organic C removal. Both MEBR and MBR generated effluents complying with local regulations at these temperatures (Fig. 2b). These outcomes indicate that such temperature decrease did not have a significant influence on C removal. The results were in agreement with a previous study (Ma et al., 2013), where the MBR attained a sufficient removal of organics at temperatures of 15 °C. Several studies also described that MBRs were not very susceptible to low-temperature reduction in terms of COD removal (Zhou et al., 2018; Chan et al., 2009; Hai et al., 2011), due to the capability of MBRs to work with high MLSS concentrations (Arévalo et al., 2014; Zhang et al., 2014; McCarty et al., 2011; Barreto et al., 2017).

In the third stage (Stage III), the addition of sludge from a reactor carrying out side-augmentation of anammox biomass (Lagum, 2019) would change the ratio of microbial community and operational conditions through which the C removal efficiency might be significantly affected. Nevertheless, the MEBR-anammox system provides a satisfactory condition for the growth of other types of bacteria including denitrifying, PAOs and floc-forming bacteria, which participate in the process of carbonaceous COD removal. Under the MEBR-anammox conditions, the competition among these bacterial consortia to break down the organic molecules as a source of energy should be very high. Furthermore, the MEBR-anammox could provide electrochemical oxidation along with biological oxidation, which resulted in higher COD removal of 92.38% (day 46) at low temperature (10 °C). By comparison, the control MBR experienced low removal of organic materials at 10 °C (average 51.58%). Similar findings were reported in the literature where recent studies showed that MBR treatment fails sharply at sewage temperatures less than 10 °C (Zhang et al., 2014; McCarty et al., 2011).

It could be concluded that electro-stimulation in addition to electro-coagulation and electro-oxidation processes as a significance of applying appropriate CD electrical field enhanced the biological activity and removal of organic matter at low temperatures, resulting in lower COD values in the MEBR-anammox process. These results are consistent with the findings of (Ibeid and Elektorowicz (2021). It was also observed that the strategic role of the CD electrical field in advancing the treatment efficiency of COD was much more evident at low wastewater temperatures than that at ambient wastewater temperatures. This could attribute to the boost of the electron transport energy and mass transfer efficiency at low temperatures. Previous batch studies confirmed the better performance of metabolism processes, biomass growth, and activity in the electro-bioreactors at low wastewater temperatures in comparison to the control bioreactors (Adam and Elektorowicz, 2018). Hence, the biological degradation of the MEBR-anammox process is further advanced as a consequence of adequate CD operation. Therefore, both biological transformation and electrochemical processes, without taking into consideration the contribution of membrane separation, resulted in a profound COD removal efficiency in the MEBR-anammox at low temperatures.

It is also noteworthy that the low DO concentrations (0.1–1 mg/L) in the MEBR-anammox (Stage III), where the system was working at DO conditions that support the anammox reaction, did not disrupt the biological treatment of COD, which is phenomenal since MBRs usually work at elevated DO levels to support aerobic treatment of COD and $\mathrm{N}{\mathrm{H}}_4^{+}$ , resulting in high power costs related to aeration (Fletcher et al., 2007).

Phosphorus removal

Temperature variations also have a significant influence on P removal processes (Ferrentino et al., 2017; Tian et al., 2013; Haiming et al., 2014). The removal of orthophosphates as a result of microbial accumulation and electrocoagulation process (Eqs 5–6) was very high in the MEBR at room temperature (Fig. 3a). The mechanism of simultaneous electrochemical and biological P removal in the MEBR system is described in Lagum (Lagum, 2021). The MBR had a very low P removal at the same operating temperature as the MEBR system. Previous studies also documented lower P removal efficiency at ambient operational temperatures in the MBR (Wei et al., 2012; Hasan et al., 2012; Zuthi et al., 2013). Stage II, operating at 15 °C, has shown almost the same removal efficiency as Stage I in terms of orthophosphates by the MEBR process. Whereas, almost complete absence of orthophosphate removal by the MBR process was observed.

In Stage III, the variation of P removal was mainly related to the change in design and operation conditions of the new MEBR-anammox, which was mostly evident in the first 20 days after the addition of anammox biomass to MEBR. In such cases, anammox microbes were dominated (71% of the reactor volume), and therefore a substantial N removal (Fig. 4f) was reached without achieving a satisfactory P removal. Restated, the key objective of this research project was to upgrade N removal under low-temperature operations. Accordingly, the priority was to adjust the conditions adequately for this purpose, and then the voltage and exposure modes can be adjusted to attain optimal COD and P removal rates.

When the technological parameter of the DC electrical field was adjusted, an immediate drop in P concentrations in the effluent was observed (Fig. 3c). Remarkably, this voltage and direct current adjustment had no negative influence on N removal. Several studies reported the effect of CD on the removal efficiency of orthophosphates (Khaled et al., 2015; Huang et al., 2016). Furthermore, the occurrence of PAO organisms at the end of the operation period was well documented by FISH technique (Lagum, 2021). This resulted in a very low ortho-P concentration in the effluent. In this stage, applying the MEBR-anammox system at low temperatures showed almost complete P removal with a minimum orthophosphate concentration of 0.1 mg\L (Fig. 3c).

It appears that the considerable P removal by MEBR-anammox was a result of the synergistic effects between bioflocs, PAO activities and electrocoagulation processes. By increasing the applied voltage and DC exposure time in the MEBR-anammox at low temperatures, more aluminum ions and more electrons were released to promote both electrochemical and biological processes; therefore, P removal was significantly enhanced. It could be indicated that the alternating operation (ON/OFF) of the electricity in the MEBR-anammox process stimulated the microbial activity responsible for P removal at low-temperature conditions.

Nitrogen removal

Nitrogen bacteria are significantly affected by temperature variations and operational conditions. N removal in the MEBR process depends on biological processes such as nitrification, anammox and denitrification. The performance of these processes is substantially influenced by the applied CD and DO levels, as well as temperature. The first stage (Stage I) operated at ambient temperature (20 ± 2 °C) witnessed outstanding $\mathrm{N}{\mathrm{H}}_4^{+}$ removal efficiencies in both reactors. However, the application of the DC electric field in the MEBR process caused an increase in $\mathrm{N}{\mathrm{H}}_4^{+}$ removal. Stage I presented an enhancement of the nitrification potential of the MEBR system compared to MBR. The enhancement in $\mathrm{N}{\mathrm{H}}_4^{+}$ removal was largely attributed to the development of a particular nitrifying community within the MEBR process. Lagum and Elektorowicz (Lagum and Elektorowicz, 2022) found the proliferation of different species of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the conventional MBR and MEBR, which subsequently led to the difference in nitrification and $\mathrm{N}{\mathrm{H}}_4^{+}$ removal performance in both types of wastewater treatment systems.

Stage II operated at 15 °C exhibited different performances in both MBR and MEBR processes. The operation of the MEBR at a temperature of 15 °C and DO of 0.5–2.5 mg\L did not significantly disturb the biological degradation of $\mathrm{N}{\mathrm{H}}_4^{+}$ via nitrifying community (≤10% decrease). Instead, these operational conditions enhanced the performance of the denitrification process beyond the nitrification, where the simultaneous removal of $\mathrm{N}{\mathrm{H}}_4^{+}$ and $\mathrm{N}{\mathrm{O}}_3^{-}$ was accomplished (Fig. 4c and 4d). The highest reduction of $\mathrm{N}{\mathrm{H}}_4^{+}$ and $\mathrm{N}{\mathrm{O}}_3^{-}$ were attained when the ORP of the MEBR fluctuates between −20 to + 126.3 mV, generating the optimum conditions for the autotrophic nitrifiers and heterotrophic denitrifiers (Eqs 7–10). These sporadic anoxic/aerobic conditions provided the lowest TN concentrations in the effluent. Nonetheless, the conventional MBR operated at high DO concentration (5–6 mg/L) was unable to decrease the ORP lower than + 100, creating only nitrification conditions.

Nitrification

Denitrification

In the MBR (Fig. 4c), the operational temperature of 15 °C led to more than 50% decrease in the nitrification efficiency when compared to the average $\mathrm{N}{\mathrm{H}}_4^{+}$ removal rate at 20 °C (Stage I). These results were in agreement with previous findings, in which reducing temperatures in conventional MBRs from 25 °C to 13 °C caused a decrease in the $\mathrm{N}{\mathrm{H}}_4^{+}$ removal efficiency by ≥ 60% (Zhang et al., 2014). Head and Oleszkiewicz (Head and Oleszkiewicz, 2004) explored the effects of a sudden decrease in temperature from 20 °C to 10 °C in SBRs where they observed a 58% reduction at 10 °C. In addition to the impact of low temperature on nitrification performance, MBR with a high DO concentration did not provide adequate conditions for $\mathrm{N}{\mathrm{O}}_3^{-}$ removal. Subsequently, TN removal in the conventional MBR was affected by both temperature and DO conditions.

It was anticipated that the system used in Stage III would advance N treatment at low temperatures due to the presence of an additional microbial culture, anammox, which called for different operational conditions (Table 2). As expected, the results showed that after a few days of operation at 10 °C, the MEBR-anammox reactor produced an effluent with a very low $\mathrm{N}{\mathrm{H}}_4^{+}$ concentration (less than 2 mg/L) (Fig. 4e). The overall $\mathrm{N}{\mathrm{H}}_4^{+}$ removal was enhanced to 94.46%, indicating anammox's advantage for ammonium reduction in low-temperature environments.

Above and beyond, anammox presence in the MEBR system enhanced TN removal efficiency by up to 88.62%. This high TN removal efficiency at low temperatures illustrates that autotrophic nitrification, autotrophic anammox and heterotrophic denitrification processes worked in harmony in the same reactor, which had to be proven in this study. Nevertheless, the MBR with a high DO concentration did not afford adequate conditions for anammox and denitrifier growth, while the attempts to drop the DO content destroyed the MBR treatment proportionally. In current wastewater treatment practices, an additional unit is required to facilitate the growth of anammox biomass. Even at low temperatures, a hybrid MEBR-anammox system allowed the co-existence of nitrifiers, anammox, and denitrifiers in the same operational unit. Nevertheless, more studies need to be performed about the resilience and recovery capacity of the proposed system.

Three microbial metabolic pathways are related to the complete conversion of $\mathrm{N}{\mathrm{H}}_4^{+}$ to N₂ gases in the MEBR-anammox (Eqs 11–13). First, AOB bacteria transform $\mathrm{N}{\mathrm{H}}_4^{+}$ to $\mathrm{N}{\mathrm{O}}_2^{-}$ under low oxygen conditions in the MEBR-anammox. Second, anammox bacteria use produced $\mathrm{N}{\mathrm{O}}_2^{-}$ with residual $\mathrm{N}{\mathrm{H}}_4^{+}$ to convert them directly to N₂ gases and some $\mathrm{N}{\mathrm{O}}_3^{-}$ ions. Third, $\mathrm{N}{\mathrm{O}}_3^{-}$ as by-products of the anammox reaction are then transformed into N₂ gases due to the activity of heterotrophic denitrifiers. Furthermore, denitrifying bacteria may use the remaining $\mathrm{N}{\mathrm{O}}_2^{-}$ or accumulated $\mathrm{N}{\mathrm{O}}_2^{-}$ caused by temperature decrease to transform them directly to N₂ gases. The activation of all diverse types of N removal bacteria in the MEBR-anammox produced effluents with a low $\mathrm{N}{\mathrm{H}}_4^{+}$ concentration of less than 1 mg/L and TN concentration of less than 10 mg/L over 60 days of operation at a temperature less than 10 °C (Fig. 4e and 4f). Contrary, in the MBR, $\mathrm{N}{\mathrm{H}}_4^{+}$ concentration was above 25 mg/L while the influent was 34 mg/L, leading to 20% $\mathrm{N}{\mathrm{H}}_4^{+}$ removal efficiency only.

In the last stage (Stage III), 5 L of anammox bacteria was transferred to the MEBR system. Furthermore, DO level was adjusted between 0.1 and 1.0 mg/L. As a result of this, the typical reddish/pink color of anammox biomass (Fig. 5) in the MEBR-anammox has changed gradually as affected by low oxygen supply to support the proliferation of nitrifying microbial communities responsible for the partial nitrification of ${\mathrm{N}\mathrm{H}}_4^{+}$ (Eq.11). During these DO conditions, a mixture of biomass including autotrophic nitrifying, heterotrophic denitrifying, and autotrophic anammox in addition to some other bacteria species (i.e., PAOs) were developed in the new MEBR-anammox, which permitted to generate a better effluent quality under low operating temperatures.

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Fig. 5

Biomass color in the MEBR-anammox system: (a) before aeration and (b) after aeration.

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Due to the complex interaction between aerobic, anoxic and anaerobic organisms, appropriate control of DO and CD electrical supplies in the MEBR-anammox system is essentially required. For instance, the air supply should meet the requirement of nitrifying bacteria but not to impede anammox and denitrifying bacteria. The aeration supply should not favor the growth of NOB as well. In this study, NOB bacteria were outcompeted at low DO conditions of the MEBR-anammox , and hence NOB bacteria are unlikely to contribute to the N removal. In addition to MEBR design, operational conditions including pH and alkalinity, the adjustment of the electrical current levels in accordance with applied temperature, activation mode of electricity (ON/OFF ratio), air distribution, and ORP levels need to be carefully selected and monitored.

The integration of an anammox process into the MEBR system substantially enhanced the removal of N compounds at low temperatures. Several recent studies have exposed the feasibility of removing nitrogen biologically at temperatures lower than 12 °C with the assistance of anammox reaction (Ma et al., 2016; Park et al., 2017; Guillén et al., 2016; Laureni et al., 2016; Zekker et al., 2016; Kouba et al., 2018; Gilbert et al., 2015). Furthermore, (Adam and Elektorowicz (2017) validated the profit of applying a bio-electrokinetic system at low temperatures to increase the biological degradation of several impurities including refractory compounds. Such enhancement in the biodegradation processes was caused by supplying an adequate sources of internal energy and electron recipients by EK processes to stimulate biological activity and metabolism at these temperatures. Subsequently, anamox combined with the other two biological processes (nitrification and denitrification) in one reactor ensured a very low concentration of TN effluent in low-temperature environments.