Introduction

In anoxic conditions, heterotrophic or/and autotrophic denitrification bacteria utilize organic carbon sources as electron donors to convert nitrite and nitrate into free nitrogen gas. A lack of organic material often slows denitrification; thus, a carbon source is added to the process to convert nitrate into nitrogen gas. However, external carbon sources in wastewater treatment plants (WWTPs) have increased the breadth of safety problems, high risk, and high operating management cost of heterotrophic denitrification associated with selecting optimal carbon sources. Instead of employing organic carbon, zero-valent iron (Fe0) is used as the electron donor. Zero-valent iron (Fe0) is a widely available, low-cost, non-toxic, easy-to-handle material (Fu et al. 2014). Many contaminants, including halogenated organics, nitrate, dyes, and phenol, have been successfully removed from groundwater and wastewater using Fe0 (Jiang et al. 2008; Siddiqui et al. 2013; Fu et al. 2014). However, Fe0 is used alone as an electron donor to remove nitrate from the environment by autotrophic and heterotrophic denitrification (Aslan and Türkman 2005; Hosseini et al. 2011; Zhao et al. 2012). As a result of the Fe0 corrosion process, H2, Fe2+, and Fe3+ are released, and hydrogen can be used as the final electron donor for heterotrophic denitrification (Zhang 2002).

However, when Fe0 is used to treat wastewater, oxidants such as dissolved O2, H2O, and NO3− consume the bulk of the Fe0 (Noubactep et al. 2009); decreasing NO3− concentration by Fe0 can represent the following Eq. (1) (Park et al. 2008):

$$\begin{array}{ccc}{4\mathrm{Fe}}^{0}+{\mathrm{NO}}_{3}^{-}+{7\mathrm{H}}_{2}\mathrm{O}& \to & {4\mathrm{Fe}}^{2+}+{\mathrm{NH}}_{4}^{+}+{10\mathrm{OH}}^{-}\end{array}$$
(1)

The nitrite formation occurs by nitrate reduction to nitrite by the iron surface (Liu and Wang 2019), as shown in Eq. (2). It is an initial chemical step to reduce nitrate when electrons transfer from the Fe0 surface to NH4+, as shown in Eq. (1), or reduced to nitrite by Fe2+, as shown in Eq. (3), or an electron donor in anoxic, or by some bacteria as shown in Eq. (3).

$$\begin{array}{ccc}{\mathrm{Fe}}^{0}+{2\mathrm{NO}}_{3}^{-}+{4\mathrm{H}}^{+}& \to & {4\mathrm{Fe}}^{2+}+{\mathrm{NO}}_{2}^{-}+{2\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(2)
$$\begin{array}{ccc}{2\mathrm{Fe}}^{2+}+{\mathrm{NO}}_{3}^{-}& \to & {\mathrm{NO}}_{2}^{-}+{2\mathrm{Fe}}^{3+}\end{array}$$
(3)

The nitrate-dependent Fe2+ oxidized (NDFO) reaction has recently been found to oxidize NO3− to NH4+ by Fe2+ in an abiotic environment (Carlson et al. 2013) or chemically oxidized to N2, as shown in Eqs. (4, 5) (S⊘ Rensen 1987), or by bacteria in the presence of hydrogen ion:

$$\begin{array}{ccc}{10\mathrm{Fe}}_{\left(\mathrm{aq}\right)}^{2+}+{2\mathrm{NO}}_{3}^{-}+{24\mathrm{H}}_{2}\mathrm{O}& \to & {\mathrm{N}}_{2\left(\mathrm{g}\right)}+{10\mathrm{Fe}\left(\mathrm{OH}\right)}_{3\left(\mathrm{s}\right)}+{10\mathrm{H}}_{\left(\mathrm{aq}\right)}^{+}\end{array}$$
(4)
$$\begin{array}{ccc}{10\mathrm{Fe}}^{2+}+{2\mathrm{NO}}_{3}^{-}+{12\mathrm{H}}^{+}& \to & {\mathrm{N}}_{2}+10 {\mathrm{Fe}}^{3+}+{6\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(5)

Consequently, through the Feammox process, Fe3+ is utilized as an electron acceptor to oxidize NH4+. The NH4+ oxidation with Fe3+ reduction (Feammox) was defined as oxidizing NH4+ to produce N2, NO2− or NO3− through reducing Fe3+ and reducing to Fe2+, as shown in Eq. (6):

$$\begin{array}{ccc}{\mathrm{NH}}_{4}^{+}+{2\mathrm{H}}_{2}\mathrm{O}+{6\mathrm{Fe}}^{3+}& \to & {\mathrm{NO}}_{2}^{-}+{6\mathrm{Fe}}^{2+}+{8\mathrm{H}}^{+}\end{array}$$
(6)

At the same time, Fe2+ is employed as an electron donor to decrease NO3−. Because Fe3+ has fewer electrons and is more stable than Fe2+, coupling of the NDFO and Feammox reactions was studied (Xu et al. 2016b), with Fe cycling used as a catalyst to decrease the need for Fe ions to prevent sludge mineralization.

However, the conversion between nitrite and Fe2+ and decreasing pH will produce N2O (Park et al. 2008), then N2O can act as an electron acceptor by electrons donated by NO3− to produce N2O and N2 following Eqs. (7–9):

$$\begin{array}{ccc}{\mathrm{NO}}_{3}^{-}+{2\mathrm{e}}^{-}+{2\mathrm{H}}^{+}& \to & {\mathrm{NO}}_{2}^{-}+{6\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(7)
$$\begin{array}{ccc}{\mathrm{NO}}_{2}^{-}+{2\mathrm{e}}^{-}+{2\mathrm{H}}^{+}& \to & {0.5\mathrm{NO}}_{2}+{1.5\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(8)
$$\begin{array}{ccc}{0.5\mathrm{NO}}_{2}+{2\mathrm{e}}^{-}+{\mathrm{H}}^{+}& \to & {0.5\mathrm{N}}_{2}+{0.5\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(9)

When Fe0 is used, the aging of Fe0 and its limited reactivity are critical issues for Fe0-based technology (Xu et al. 2016b). Therefore, finding practical ways of significantly increasing Fe0 reactivity is crucial. Many researchers employed various technologies to enhance Fe0 reactivity, such as ultrasonic, acid washing, H2-reducing pretreatment (Lai and Lo 2008), electrochemical shorthand (Chen et al. 2012), Fe0-based bimetals (Lim et al. 2007), and nanosized Fe0-nFe0 (Huang et al. 2013). These technologies are always complicated, costly, and hazardous to the environment (Jiang et al. 2015). As a result, other technologies for increasing the reactivity of aging Fe0, such as pre-magnetization or applying a weak permanent magnetic field (WMF) with Fe0, were used to eliminate high concentrations of p-nitrophenol (PNP), SO2 removal (Jiang et al. 2008; Siddiqui et al. 2013), accelerate chloroacetamide removal from drinking water, enhance phenol degradation, partial nitrification, CH4 production, triethyl phosphate degradation, and antibiotic degradation (Wang et al. 2015; Wang et al. 2017; Chen et al. 2019; Huang et al. 2019; Pan et al. 2019, et al. 2019).

Additionally, several studies have found that the effects of permanent magnets or electromagnets can change water pH, oxidation–reduction potential (ORP) (Yin et al. 2011; Hassan and Rahman 2016), increase electron density, and promote electron transfer for redox reactions (Yap et al. 2021). The magnetic field mainly affects the material’s properties, structure, photocatalysis, electrodynamics, synthesized reaction, isomerization reaction, nuclide enhancement reaction, increasing electron density in water, and metabolic reaction (Hassan and Rahman 2016). Coupling magnetic field (MF) with Fe0 is chemical-free (Xu et al. 2016a); it contributes to the release of Fe2+ from the Fe corrosion process (Li et al. 2017b; Ren et al. 2018), which can significantly improve the pollutants’ targeting, reduce Fe0 doses, extend the operating pH range (Sun and Guan 2019; Wang et al. 2020b), and give energy to the donor electron (Salehani et al. 2010), and influence the anions’ movement simultaneously with paramagnetic Fe2+ to keep electroneutrality (Sun and Guan 2019). The theoretically essential point of coupling electric–magnetic (MF) with Fe0 in this study is to promote the increase of Fe2+, accelerating the Fe3+ reduction to Fe2+ because the reduction rate of Fe3+ is much slower than the oxidation rate of Fe2+ because the concentration of Fe2+ would decrease rapidly during pollutants’ depredation (Chu et al. 2021).

However, many researchers are working to achieve a low dose of Fe2+ with effective pollutant degradation and removal from wastewater (Taherdanak et al. 2016). Fe2+ will promote decreasing pH and increase the reaction rate of non-target substrates (H+ and O2) with Fe0 and would undoubtedly increase the Fe0 corrosion rate concomitantly with pollutants (Guan et al. 2015). Therefore, most bacteria species use Fe2+ as the electron donor for metabolism (Straub et al. 2004), promoting microbial abundance diversity and increasing bacteria’s metabolism by promoting Fe2+ (Li et al. 2017a). Anaerobic denitrifying Fe2+ oxidation bacteria grow by using Fe2+ as a source of energy and electron donor in marine environments or freshwater with a narrow pH range to 7 (Kappler et al. 2005; Hedrich et al. 2011). An essential point in this study is to enhance nitrogen removal efficiency in the upflow microaerobic sludge reactor (UMSR) by coupling the electric–magnetic field (MF) with Fe0. By coupling the electric–magnetic field (MF) with Fe0, there is a possibility of continuously releasing H2, Fe2+, and Fe3+ from the Fe0 corrosion process to give activity to some heterotrophic nitrifying bacteria and denitrifying bacteria that depend on Fe2+ and Fe3+, or H2 for metabolism in the absence of the organic carbon source. Additionally, coupling the electric–magnetic field (MF) with Fe0 may prevent Fe3+ precipitation on the bacterial cell surface by chemically reducing Fe3+ to Fe2+. This research aimed to study a novel upflow microaerobic sludge reactor (UMSR) operated for 78 ± 2 days continuously, under five operating stages with and without coupling electric–magnetic field (MF) with Fe0. At the same time, the NH4+-N, NO3−-N, NO2−-N, TN removal, and microbial community structure were evaluated in this research.

Methods and materials

As shown in Fig. 1, the UMSR has an inner volume of 4.9 L, and the HRT was 2.5 h; the influent was injected from the bottom of the reactor. A reflux aeration tank controlled dissolved oxygen (DO) in the reactor was held at less than 1.0 mg/L by a reflux aeration tank. An extra heating system maintained the temperature at 25–27 °C by a supplementary heating system. The electric–magnetic field of 40 mT was adjusted by 6 V–50 Hz alternate current (AC) and put deep into the sludge. The UMSR continuously operated for 78 ± 2 days, divided into five operating stages of 15 ± 1 days for each stage (without Fe0, with Fe0, coupling with MF, without coupling with MF, and coupling with MF again) until the Fe0 corrosion was completed.

Fig. 1
figure 1

Schematic diagram of USMR setup

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Synthetic nutrients and sludge

The nutrients in this synthetic wastewater were composed of (mg/L): NH4Cl (190), KH2PO (25.2), KHCO3 (124.8), CaCl2 (300), and MgSO4 (200). The inoculated sludge was taken from the Taiyuan wastewater treatment plant, with the mixed liquor suspended solids (MLSS) and the mixed liquor volatile suspended solids (MLVSS) being 2.42 mg/L and 1.936 mg/L, respectively. The ratio of Fe0 powder and Fe–C powder was 3:1.

Analytical methods and equipment

Before experimenting with new conditions, the reactor was operated at its optimized condition for more than 360 days at 25–27 °C. An external heating wire and a temperature controller are used to regulate the inside temperature of the reactor (XH-W2140, China). The pH and temperature were measured using pH and temperature meters (Hanna Instruments CAL Check™ HI5221). The concentrations of NH4+-N, NO3−-N, NO2−-N, and TN in effluent were measured daily using standard methods (Association 1915).

Microbial community analysis

The sludge samples were taken from three continual operation conditions (before adding Fe0, after adding Fe0, and after coupling MF with Fe0) labeled as (S1, s2, and s3) to identify the microbial community structure during that three operating conditions. They were sent to Shanghai Lingen Biological Technology Co., Ltd to analyze the microbial diversity. After completing the genomic DNA extraction, genes were amplified using the specific primer with the barcode, 16S V4-V5: 515F–907R, 18S V9: 1380F–1510R, ITS1: ITS1F–ITS2R. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and the Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina MiSeq platform, and 250 bp/300 bp paired-end reads were generated.

Results and discussion

Parameters with operating conditions and reactor performance

Table 1 summarizes the performance of the UMSR and coupling electric–magnetic field (MF) system concerning the NH4+-N, NO3−-N, NO2−-N, and TN removal for each stage of operation. For these data, the process continued for 78 ± 2 days until the Fe0 was corroded under an anaerobic condition with electric–magnetic field effects.

Table 1 Parameters and operating conditions
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NO3 −-N removal efficiency

As shown in Fig. 2, the concentration of NO3−-N in the effluent dropped quickly for the first few days after Fe0 was added to the reactor. After a few days, the concentration of NO3−-N increased again. After two periods of coupling MF with Fe0 (first coupling with MF and second coupling with MF), the NO3−-N concentration dropped to a stable level. The NO3−-N removal efficiencies were 58% before adding Fe0, 64% after adding Fe0, 76% at first coupling with MF, 55% without coupling MF, and 76% at second coupling with MF, respectively. The highest and most stable removal was observed during MF coupled with Fe0. Coupling Fe0 with electric–magnetic provides stability for nitrate removal by producing H2 and Fe2+. It is one of the factors that donate to denitrification by Fe0 corrosion. It can also happen that hydrogen can be used as an electron donor because of the corrosion of Fe0 in a magnetic field (Liu and Lowry 2006; Němeček et al. 2014). The magnetic field affects electrode kinetics by the accelerated effect on the cathodic reaction of the single Fe0 corrosion process. It will affect the Fe3+ reduction to Fe2+ in this Fe0 recycling. It is possible for nitrate-dependent Fe2+ oxidized (NDFO) to occur by producing more Fe2+ because the reductive efficiency of nitrate by Fe0 increased by the increase in dissolved ferrous ion concentration (Fe2+aq). It may also release cathodic H2 by reducing water-derived protons (Weathers et al. 1997), as shown in Eqs. (10, 11).

Fig. 2
figure 2

Effluent pH and NO3−-N concentration

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$$\begin{array}{ccc}{\mathrm{Fe}}_{\left(\mathrm{s}\right)}^{0}+{\mathrm{H}}_{2}\mathrm{O}& \to & \mathrm{Fe }{\left(\mathrm{OH}\right)}_{2\left(\mathrm{aq}\right)}+{\mathrm{H}}_{2\left(\mathrm{g}\right)}\end{array}$$
(10)
$$\begin{array}{ccc}{\mathrm{Fe}}_{\left(\mathrm{s}\right)}^{0}+{2\mathrm{H}}^{+}& \to & {\mathrm{Fe}}_{\left(\mathrm{aq}\right)}^{2+}+{\mathrm{H}}_{2\left(\mathrm{g}\right)}\end{array}$$
(11)

As a favored electron donor, hydrogen gas produced by Fe0 corrosion can be an advantage for several metabolic groups of denitrifying bacteria. The Fe2+ oxidation rate could influence various abiotic factors, including a concentration in anionic species; for instance, concentration in anionic species such as Cl− in water will reduce the Fe2+ oxidation rate and laded to a higher accumulation of Fe2+ (Adeleye et al., 2013). The oxidation of Fe2+ increases the hydrogen ion H+ concentration in the water, which reflects on the pH, as shown in Eqs. (4, 6). The pH is an important parameter that strongly affects the nitrate reduction by Fe0 because the nitrate requires proton participation, which affects Fe0 corrosion rate and Fe corrosion product, and Fe2+ occurs at lower pH (Weathers et al. 1997). It was decreased at the first stage of adding Fe0 to the reactor. The primary way to reduce NO3− is by Fe0; the Fe0 acts as an electron donor to reduce NO3− by Fe0 surface. The nitrate reduction depends on electron transfer efficiency from the Fe0 surface Fe0 → Fe2+  + 2e−.

The existence of Fe0 and Fe2+ may involve other reactions. They are sources of electrons and protons, as shown in Eqs. (12, 13):

$$\begin{array}{ccc}{3\mathrm{Fe}}^{0}+{4\mathrm{H}}_{2}\mathrm{O}& \to & {\mathrm{Fe}}_{3}{\mathrm{O}}_{4}+{8\mathrm{H}}^{+}+{8\mathrm{e}}^{-}\end{array}$$
(12)
$$\begin{array}{ccc}{3\mathrm{Fe}}^{2+}+{4\mathrm{H}}_{2}\mathrm{O}& \to & {\mathrm{Fe}}_{3}{\mathrm{O}}_{4}+{8\mathrm{H}}^{+}+{2\mathrm{e}}^{-}\end{array}$$
(13)

Hematite (Fe2O3) and magnetite (Fe3O4) were discovered in these probable reactions, as shown in Eqs. (14, 15). It would play a part in the pH decreasing due to Fe0 corrosion serving as an electron donor from the Fe0 surface Fe0 − 2e → Fe2+.

$$\begin{array}{ccc}{2\mathrm{Fe}}^{0}+{3\mathrm{H}}_{2}\mathrm{O}-{6\mathrm{e}}^{-}& \to & {\mathrm{Fe}}_{2}{\mathrm{O}}_{3}+{6\mathrm{H}}^{+}\end{array}$$
(14)
$$\begin{array}{ccc}{3\mathrm{Fe}}^{0}+{4\mathrm{H}}_{2}\mathrm{O}-{8\mathrm{e}}^{-}& \to & {\mathrm{Fe}}_{3}{\mathrm{O}}_{4}+{6\mathrm{H}}^{+}\end{array}$$
(15)

Moreover, the nitrate reduction with H+ will be consumed at neutral pH:

$$\begin{array}{ccc}{\mathrm{NO}}_{3}^{-}+{10\mathrm{H}}^{+}+{8\mathrm{e}}^{-}& \to & {\mathrm{NH}}_{4}^{+}+{3\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(16)

When magnetic coupling in all phases, the lowest pH was at the first operating conditions when MF coupling with Fe0. The pH value decreased to 5.2 at the first stage of coupling MF with Fe0. The decreasing pH was low and the most stable at all stages of coupling MF with Fe0, as shown in Figs. 2, 3, and 4.

Fig. 3
figure 3

Effluent pH and NH4+-N concentration

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Fig. 4
figure 4

Effluent pH and NO2−-N concentration

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NH4 +-N removal efficiency

The effluent NH4+-N concentration increased significantly from 21 to 35 mg/L in the first 4 days after Fe0 was added to the reactor, followed by a decrease in NO3−-N concentration from 11 to 6 mg/L. Because the reaction between Fe0 and NO3− converts NO3− to NH4+, as shown in Eq. (1), it increases the NH4+ concentration. The nitrogen removal efficiency could not increase during the first few days after Fe0 was applied to the reactor. It was noticed that the highest NH4+-N removal occurred during stage (3) when MF was first coupling with Fe0. The coupling of NH4+ oxidation to NO2− with Fe3+ reduction is possible, as shown in Eq. (6). After a few days, the NH4+-N concentration became lower day by day and stable, as shown in Fig. 3; the NH4+-N removal efficiencies were 47% before adding Fe0, 56% after adding Fe0, 72% at the first coupled MF with Fe0, 74% without coupling with MF, and 75% at the second coupled MF with Fe0, respectively. Thus, the NO2− produced from NH4+ oxidation with Fe3+ will be involved in the ANAMMOX reaction of ammonia, as shown in Eq. (17):

$$\begin{array}{ccc}{\mathrm{NH}}_{4}^{+}+{\mathrm{NO}}_{2}^{-}& \to & {\mathrm{N}}_{2}+{2\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(17)

NO2 −-N removal efficiency

The NO2−-N concentration increased to 2.3 mg/L during stage (2) of adding Fe0 to the reactor due to nitrite formation caused by nitrate reduction, as shown in Eqs. (2, 3) or by NH4+ oxidation with Fe3+ (Feammox) process, as shown in Eq. (6), whereas the NO2−-N concentration before adding Fe0 was 0.1 mg/L, 0.5 mg/L at first coupled MF with Fe0, 0.7 mg/L without coupled MF, as shown in Fig. 4. Thus, the ANAMMOX reaction that makes increases NO2.− concentration, as shown in Eq. (18)

$$\begin{array}{ccc}{2\mathrm{NH}}_{4}^{+}+{3\mathrm{O}}_{2}& \to & {2\mathrm{NO}}_{2}^{-}+{4\mathrm{H}}^{+}+{2\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(18)

While the Fe3+ serves as an electron acceptor, Fe3+ oxidized NH4+ into NO2− and NO3−, and the effluent’s NO2−-N concentration was accumulated. On the other hand, by accumulated NO2−, the ANAMMOX reactions with NH4+ and NO2− play a part in the process of reducing NO2−, as shown in Eq. (19):

$$\begin{array}{ccc}{\mathrm{NH}}_{4}^{+}+1.32 {\mathrm{NO}}_{2}^{-}+0.066 {\mathrm{HCO}}_{3}+0.13 {\mathrm{H}}^{+}& \to & 1.02 {\mathrm{N}}_{2}+0.26 {\mathrm{NO}}_{3}^{-}+0.066{\mathrm{CH}}_{2}{\mathrm{O}}_{0.5}{\mathrm{N}}_{0.15}+2.03 {\mathrm{H}}_{2}\mathrm{O}\end{array}$$
(19)

As well, the electron donated by NO3− or which is released by Fe0 surface with a high concentration of H+, the NO2− will act as an electron donator to produce N2O, and then N2O will serve as an electron acceptor to produce N2, as shown in Eqs. (7–9), which gives removal stability and a decrease in NO2− accumulation. Moreover, other research has shown that the adsorption of Fe2+ on iron oxides (e.g., Fe3O4, FeOH, –FeO(OH)) is crucial in decreasing NO3−, NO2−, and NH4+. Fe2+ ions ionize H+, which may be adsorbed on the surface and reduced to active NO* by hydrogen produced by Fe corrosion after reducing NO3− to NO2−. This approach was not discussed in this work regarding electric–magnetic field effects.

Structure and function of the microbial community in the reactor

As shown in Fig. 5a, b, microbial community analysis detected that relative abundance changed at the levels of phyla and genus due to the changes in conditions. (The relative domain abundance > 1%). A notable increase and changes in diversity and a decrease in certain bacteria were detected in all samples associated with the three operating conditions. After adding the Fe0 and coupling it with the magnetic field, there were changes in certain bacteria’s richness, abundance, and inhibition.

Fig. 5
figure 5figure 5

a Relative abundance by phylum. b Relative abundance by genus

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At the phylum level, the most bacteria abundance of nitrogen removal was identified, including nitrifiers, denitrifiers, and anammox bacteria. Proteobacteria were the most dominant phylum in S1 of which 56% are known for their metabolic diversity and variety including denitrifiers, which are used in crucial environmental cycles such as carbon, nitrogen, sulfur, and phosphorus (Friedrich, Bardischewsky et al. 2006; Meier et al. 2016) and the detected genera in S1 are Rhodanobacter of which 16% is capable under acidic and anaerobic conditions of producing N2 and N2O by using nitrate, nitrite, and nitrous oxide as electron acceptors (Van Den Heuvel et al. 2010) and Denitratisoma of which 14% is anaerobic oxidative that reduces nitrate to N2O and N2 and may lead to nitrite accumulation with some N2 fixation strains (Fahrbach et al. 2006); 45 of SWB02 and 2% of Ellin6067 oxidize nitrites to nitrates (NOB) (Fumasoli et al. 2015); 4% of Thermomonas, sulfur oxidizers, and carbon fixing convert CO2 to carbonate, where nitrogen fixation is related (Castelán-Sánchez et al. 2020); 3% of Limnobacter is heterotrophic sulfur oxidation and may adapt with anaerobic methane oxidizer bacteria (AOM) (Chen et al. 2016), and 1% of Thiobacillus obtains energy from oxidation and reduction of ferrous and sulfide. Moreover, Thiobacillus reduces nitrate, nitrite, and other nitrogen compounds to dinitrogen (Kelly and Wood 2000).

However, a sharp decrease in the level of the Proteobacteria was noted after adding the Fe0 to the reactor, while richness in diversity and diminishing were observed in both samples s2 and s3, at the level of genus Stenotrophomonas, which can use nitrate as a terminal electron acceptor for growth with oxygen absence (Crossman et al. 2008) of 4% in s2, Brevundimonas which have potential of N2 fixation (Jiang et al. 2022) of 2% in s2, Diaphorobacter under anaerobic conditions reduces nitrate and nitrite to N2 (Qiu et al. 2015) of 1% in s2, Qipengyuania of 1% in s2 has some strains that reported can participate in biogenic nitrogen cycling (Liu et al. 2022), Pseudoxanthomonas of 2% in s2 and s3 it is anaerobic methane oxidation bacteria (AOM) it can utilize methane as electron donor to reduce NO2−, NO3−, Fe3+, and SO42−, it typically require additional electron donors such as hydrogen (Fu et al. 2019), Hydrogenophaga of 4% in s3 it is capable nitrate denitrification and using the oxidation of H2 as an energy source and CO2 as a carbon source (Iannacone et al. 2020; Xu et al. 2021), Pseudomonas of 6% in s2, 4% in s3 it has been reported that hydrocarbon-degrading bacteria and may provide a carbon source and energy to other bacteria (Wang et al. 2020a), Acinetobacter of 4% in s2, 2% in s3 which use Fe2+ as electron donor be capable of denitrifying (Kiskira et al. 2017), the Limnobacter of 2% in s2, and of 3% in s3, SC-I-84_norank of 1% in s2 and s3, Steroidobacteraceae_uncultured was of 1% in s2, 2% in s3, the Thermomonas was of 1% in both samples s2 and s3, and Xanthobacteraceae_uncultured of 1% in s3, respectively.

Meanwhile, the nitrifiers (AOB) detected in the reactor included Nitrospirota and Bacteroidetes; these nitrifiers bacteria are often present in activated sludge. The Nitrospirota was 5% in S1 and was shared by the genus Nitrospira of 5%. It is a chemolithoautotrophic nitrite-oxidizing bacterium and is essential for nitrification in the completed nitrogen cycle when it oxidizes nitrite to nitrate from ammonia (Yang et al. 2022). However, the Nitrospira may also give ammonia oxidizers released from urea or cyanate in interactions known as “reciprocal feeding.” Recently found members of the Nitrospira can catalyze both nitrification stages independently, earning them the name complete ammonia oxidizers or “Comammox” bacteria (Koch et al. 2019). Certain Nitrospira strains may also use hydrogen and formate to supplement their aerobic nitrite oxidation with oxygen or nitrate as the terminal electron acceptor (Palomo et al. 2018; Koch et al. 2019). The Nitrospira can adapt to a wide range of oxygen conditions according to its metabolic system (Mehrani et al. 2020) as well as observe that at the level of Chloroflexi, it was not the most dominant bacteria in sample S1. Thus, after the Fe0 was added or coupled with MF, Chloroflexi became one of the most predominant bacteria at 21% in S2 and 28% in S3, while it was 2% in S1. It was reported that chemoautotrophic desulfurization and denitrification of bacteria and one of the heterotrophic bacteria derive energy from H2. It was found in low-pH soils with carbon dioxide fixation (Long et al. 2015; Islam et al. 2019). At the genus of desulfurization and denitrification bacteria SBR1031_norank of 13% in s2 and 18% in s3, heterotroph Anaerolineaceae_uncultured was 1% in s2 and 2% in s3. Chemolithotroph-denitrifying bacteria use Fe2+ as electron donor, OLB14_norank of 1% in s2 and s3, and KD4-96_norank by 1% in both samples s2 and s3, respectively. This can be explained by the effect of the magnetic field on Fe0 erosion and releasing more hydrogen and ferrous ions that enter into its metabolism process, as well as possibly due to the richness of Firmicutes and hydrogen production in this condition. However, Firmicutes were absent before adding the Fe0 to the reactor. It has been reported that when coupled with iron, it generates hydrogen (Mohan et al. 2011). Before adding Fe0, Firmicutes were absent in sample S1; in contrast, after adding Fe0 and coupling with MF, the abundance was 4% in s2 and 5% in s3, respectively, at the genus Lysinibacillus of 1% in both samples s2 and s3. This can be explained by some Firmicutes strains exploiting acids as a source of carbon and energy; the decrease in pH and the existence of Fe0 provided the richness of the Firmicutes bacteria in the reactor.

Whilst, before adding Fe0 to the reactor in sample S1, the Gemmatimonadota phylum of 6%, on the level of the genus Gemmatimonadaceae_uncultured of 6%. The Gemmatimonadota proportion decreased to 3% after adding Fe0 in s2, and after coupling with MF, an enrichment of 4% was observed in s3, shared by the same genus Gemmatimonadaceae_uncultured. In contrast, the Gemmatimonadota groups have been found in various ecosystems and activated sludge used in wastewater treatment. These groups seem to depend on urea hydrolysis inside cells for energy (Chen et al. 2021; Mujakić et al. 2022). Anammox bacteria Planctomycetota of 4% in S1, 3% in s2 and s3. Planctomycetota in S1 was shared by genus 2% of Schlesneria, which utilizes an organic nitrogen compound as nitrate and ammonium as nitrogen sources; after adding Fe0 and coupling with MF, that genus vanished in both samples s2 and s3. After adding Fe0 to the reactor and coupling with MF, the genus Saccharimonadales_norank has increased by 1% in s2 and 2% in s3 and noted that the genus Patescibacteria increased by 2% in s2 and 3% in s3, while it was 1% in S1, respectively.

In the same context, 1% of Armatimonadota in S1 and 2% in s2 and s3 has been shared by genus Armatimonadota_norank by 1% in S1 and 2% in samples s2 and s3, respectively. Furthermore, Actinobacteriota was absent in sample S1 and appeared after Fe0 was added in s2 and s3 by 6%, with genus 67-14_norank in both samples s2 and s3 by 2%. Actinobacteriota have some strains that produce organic acids and siderophores which might delay the biogenic Fe2+ reoxidation and decrease pH (Zhang et al. 2019). In anoxic conditions, many Actinobacteriota reduces Fe3+ in the iron cycle and plays as main important bacterium in the denitrification process (Huang et al. 2022). After adding Fe0, Deinococcota abundance was increased and returned to the same proportion after coupling with MF at the same genus Meiothermus by 1% in S1, 2% in s2, and 1% in s3, which is chemoorganoheterotroph and use nitrate as the terminal electron acceptor (Song et al. 2021).

Conclusions

Adding Fe0 and coupling it with the electric–magnetic was favorable for large-scale applications due to its simple operation, low cost, and high effectiveness for nitrogen removal. As a result, Fe0 corrosion under an electric–magnetic field may provide Fe2+ and Fe3+ and be used as an electron donor to oxidize NO3− to NH4+. By producing H2 and decreasing pH and using it as an electron donor donated by NO3− to produce N2O and N2, the NH4+-N, NO3−-N, and TN removal efficiencies in the UMSR are enhanced under MF with Fe0. Through coupling electric–magnetic with Fe0, the energy would be served to electron donors from the Fe0 surface and increase electron density. The direction of electron transfer from Fe0 to nitrate would be directly converted into N2; on the other hand, NO3− converted into NH4+ and NO2− before the coupling MF stage.

Moreover, adding Fe0 and coupling with electric–magnetic to the reactor decreased the level of Proteobacteria and Nitrospirota. Changing microbial communities became the dominant abundance besides the Proteobacteria, such as Chloroflexi and Firmicutes. After Fe0 was coupled with electric–magnetic, it gave richness to some anammox such as Saccharimonadales_norank and Patescibacteria. Denitrification bacteria depend on ferrous Fe2+ as the source metabolisms, such as Anaerolineaceae_uncultured and Acinetobacter, and ferric iron Fe3+ such as Pseudoxanthomonas, or use hydrogen ion such as Hydrogenophaga. However, some bacteria genera have been found with an apparent richness, and the functions have not been determined by researchers yet.