Effect of magnetic fields on simultaneous nitrification and denitrification microbial systems

The accumulation of nitrogen-containing compounds in the aquatic environment leads to the serious deterioration of water quality, such as by eutrophication, resulting in the death of a large number of organisms due to aquatic hypoxia, which not only affects economic benefits but also may threaten human health (Guerrero et al. 2016; Zhang et al. 2022a).

Microbial systems with excellent catalytic properties have been well studied as catalysts because of their unique physicochemical behavior (Ferreira Mota et al. 2022; Brandão Júnior et al. 2023). Microbial systems are remarkably effective in the versatile reactions, such as hydrolysis, esterification, transesterification, alcoholysis, and C–C bond formation (Virgen-Ortíz et al. 2019). Microbial systems have been recently used as a potential biocatalyst in a large number of biotechnological sciences; more specifically, these include dairy products (cheese recovery, flavor enhancement and the production of enzyme-modified cheese (EMC), detergents, pharmaceuticals (ibuprofen, naproxen), chemicals, agriculture products (pesticides, insects), and oil chemistry (fat and oil hydrolysis, and the synthesis of bio-detergents) (Villalba et al. 2016; Moreira et al. 2020; Rodrigues et al. 2023). Microbial systems act as a good catalyst; therefore, its production and utilization may be a better alternative of chemical catalysts (dos Santos et al. 2014; de Sousa et al. 2023).

For nitrogen removal, traditional nitrification and denitrification reactions are carried out in different microbial systems. Simultaneous nitrification and denitrification (SND) refers to the simultaneous occurrence of two processes (nitrification and denitrification) in one reaction vessel to realize the removal of nitrogen from sewage. Compared with the traditional biological denitrification process, SND is carried out with a small volume and short reaction time, which can effectively reduce operating costs and energy consumption (Bhattacharya & Mazumder 2021). Zeng et al. (2004) found that SND removes 80–96% of total nitrogen and requires fewer external carbon sources and alkalis, reducing sludge production by 30% (Seifi & Fazaelipoor 2012). Particularly, SND bacteria possess better nitrogen removal performance and faster growth rate that can utilize various carbon sources, making them gradually become dominant bacteria (Yan et al. 2022). To date, SND has been applied in various processes including sequencing batch reactors, moving bed biofilm reactor, and membrane bioreactor. The symbiotic coexistence of aerobic nitrifying bacteria, anaerobic denitrifying bacteria, and facultative microorganisms is the key to the stable operation of SND systems, and it is also a popular topic in SND technology research. The significance of symbiosis is that different microbial populations can establish connections more quickly with regard to metabolic pathways, such as ammonia oxidation and nitrate/nitrite reduction, which is beneficial for the biological process of SND (Guerrero et al. 2016). Layer et al. (2020) found that large-sized activated sludge was more conducive to the coexistence of microorganisms. Sludge granulation limits the diffusion and mass transfer of dissolved oxygen (DO). A gradient of DO was formed on the surface of microbial flocs, forming aerobic and anoxic regions from the outside to the inside. The extracellular polymeric substances (EPS) secreted by microorganisms were conducive to the formation of granular sludge, which in turn formed bacterial micelles, facilitating the coexistence of microorganisms. The above studies provide references and lessons for the development of SND technology. However, a low microbial growth rate and difficulty in regulating aerobic and anoxic environmental conditions in a single reactor (Bhattacharya & Mazumder 2022) hinder the application of SND technology.

The magnetic fields within proper ranges can increase the flexibility of nitrification and denitrification processes, promote the enzyme activity, and significantly facilitates the bioreaction process and the microbial growth and metabolism, which, as a result, improve the relative abundance of bacteria and genes relating to nitrogen removal (Dixon & Abbas 2016). It has been found that magnetic fields with certain strength can stimulate the secretion of extracellular polymers, effectively accumulate metal ions, and reduce the zeta potential of extracellular polymers, which is conducive to inducing adhesion and aggregation between particles to form large sludge particles (Wang et al. 2012; Yan et al. 2015). In addition, magnetic fields can promote nitrogen compound conversion and ammonium-oxidizing bacterial (AOB) activity, thereby enhancing the nitrification rate (Wang et al. 2012). Liu et al. (2022) found that a 50 mT magnetic field enhanced the nitrosation and denitrification of AGS, and the removal rates of ammonia nitrogen, total nitrogen, and chemical oxygen demand (COD) increased by 26.2, 41.5, and 7.6%, respectively, compared with those of the nonmagnetic control group. Xu et al. (2020) found that a magnetic field strength of 30 mT improved the enzyme activity and the relative abundance of bacteria and genes, and the removal rates of total nitrogen and ammonia nitrogen increased by 22.4 and 39.5%, respectively. On the other hand, the positive effects by magnetic fields occurred in suitable strength ranges. Magnetic fields with too strong or weak strength may limit biological activity. In addition, the differential facilitation effect of magnetic fields on different bacteria could potentially alter the balance of the original microbial structure and thus affect the nitrogen removal performance in bioreactor (Wang et al. 2021). These studies promoted future researches on the application of magnetic fields for enhanced nitrogen removal in wastewater treatment. However, for SND systems, the positive effects and mechanisms of magnetic fields still need further exploration.

Therefore, in this study, a magnetic SND (MSND) reactor was constructed by electrifying a coil. The reactor was running sequentially in batches which included the inlet, aeration, precipitation time, and drainage. The effects of optimized magnetic field (0, 10, 20, and 30 mT) strength on nitrogen removal performance, the structure characteristic, and predicted functions of the microbial community were investigated and illustrated through the spectroscopy, high-throughput sequencing, and predicted metagenome. Therefore, the mechanisms of enhanced nitrogen removal in MSND was comprehensively explored.

The reactor was made of plexiglass and had an effective volume of 2 L. The reactor was equipped with a coil winding, and 12 iron cores were used (120 rpm) instead of employing mechanical stirring, which could improve the matrix transport and the utilization capacity by the microbial community. By adjusting the coil current, four kinds of reactors with different magnetic field strengths were constructed, namely, reactor a (0 mT), b (10 mT), c (20 mT), and d (30 mT) as shown in Figure 1. The reactor was running sequentially in batches (6 h per cycle), the inlet time was 5 min, aeration was performed at 30 min intervals (300 min), the precipitation time was 50 min, the drainage time was 5 min, the hydraulic retention time (HRT) was 16 h, and the operating temperature was 28.0–30.0 °C. For aeration, oxygen was provided by an air pump and microaeration sand head, and a gas rotameter was used to adjust the aeration volume such that the DO in the reactor was maintained at approximately 1.0 mg/L, the pH was maintained at 7.0–8.0, and the inlet, outlet, and aeration were automatically controlled by the timing switch. The microbial system was not a commercial. The inoculated sludge was taken from the sludge of the secondary sedimentation tank of a water plant in Jinan City, and the sludge concentration after dilution was approximately 4,000–5,000 mg/L. This test was run for approximately 45 days, the start-up stage was approximately 30 days, and daily regular sampling was performed, mainly for -N, -N, -N, and TN. The methods for water quality analysis are shown in Table 1.

Table 1

Analytical methods for the detection of -N, -N, -N, and TN

Water quality index .	Analytical method .
-N	N-(1-naphthol)-ethylenediamine photometry
-N	Nath's reagent spectrophotometry
-N	Ultraviolet spectrophotometry
TN	Alkaline potassium persulfate oxidation spectrophotometry

Water quality index .	Analytical method .
-N	N-(1-naphthol)-ethylenediamine photometry
-N	Nath's reagent spectrophotometry
-N	Ultraviolet spectrophotometry
TN	Alkaline potassium persulfate oxidation spectrophotometry

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The influent of the reactor was simulated municipal sewage; NH₄Cl and CH₃COONa provided -N and carbon sources (COD/TN = 4.0), respectively, and NaHCO₃ was used to adjust the pH value. The synthetic wastewater included CH₃COONa (COD 200 mg/L), -N (50 mg/L), CaCl₂ (20 mg/L), MgSO₄·7H₂O (20 mg/L), NaHCO₃ (500 mg/L), and trace elements 1 mg/L (Leachate collected from garden soil).

Based on the Illumina NovaSeq sequencing platform, the library was double-end sequenced (Paired_End) to study the 16S rRNA gene in the V3–V4 region amplified by 16S coding region-specific primers (16S V4: 515F-806R). The sludge was amplified and sequenced after SND was successfully started and stably operated for 14 days, and the primer sequences of the polymerase chain reaction (PCR) region were 806R (GGACTACNNGGGTATCTAAT) and 341F (CCTAYGGGRBGCASCAG). After read splicing and filtering, operational taxonomic units (OTUs) were clustered for species annotation and abundance analysis. Using R version 4.1.0 for principal coordinate analysis (PCoA), according to the KEGG database and PICRUSt analysis method of gene function prediction, the metabolic function information of different microbial communities was estimated.

According to the analysis shown in Figure 2(3) and 2(4), during the start-up process, different magnetic field strengths had different effects on nitrification and denitrification, among which reactor (d) with a 30 mT magnetic field strength showed an inhibitory effect on the nitrite-oxidizing bacteria (NOB) flora, the nitrous nitrogen effluent was unstable, and the effluent concentration was relatively high. The effluent concentration of nitrate nitrogen in the 0 mT reactor (a) was relatively high, and the efficiency of the denitrification process was relatively poor, which may have been caused by insufficient carbon sources (Qin et al. 2005). The nitration reactions in the 0 mT reactor (a) and the 30 mT reactor (d) were relatively fast, and it was speculated that a portion of the carbon source may have been consumed in the nitrification process. Later, a comparison of nitrogen concentrations in different reactors revealed that the SND start-up was successful.

From the first aeration interval to the end of a cycle, the ammonia oxidation process of the 30 mT reactor (d) was fast, and the ammonia nitrogen concentration in the effluent was the first to be reduced to the minimum value; the nitrous nitrogen oxidation process of the 0 mT reactor (a) and the 30 mT reactor (d) was faster, and the nitrous nitrogen concentration was the first to be reduced to the minimum value. It can be seen that in the whole reaction cycle, the speed of nitrogen transfer in each stage was different, and in the denitrification process, the nitrate nitrogen concentration in the magnetized reactor was consumed quickly, which may have indicated that the presence of a magnetic field effectively balanced the utilization efficiency of microorganisms in the reactor for carbon source utilization and improved the nitrogen removal effect (Hou et al. 2020). The maximum nitrogen removal performance reached 89.60% in the magnetic field of 20 mT, and the maximum nitrogen removal load reached 0.18 kg N/(m³·day). Furthermore, in the presence of magnetic fields, the total nitrogen concentration was lower than that in the control reactor overall, which is consistent with Chen et al.’s (2022) research.

The original sludge sample R before dilution and sludge samples a, b, c, and d of the four reactors after the SND system was successfully started and stabilized for 14 days were taken for diversity analysis. In this experiment, Chao1, Shannon, ACE, and Goods coverage were selected among several different alpha diversity indices to analyze the diversity and richness of the microbial communities in the sample and to visualize the sequencing depth and data volume, as shown in Table 2. In this study, the Goods coverage index was high, indicating that the probability of the sequence not being detected in the sample was very small and that it could realistically reflect the microbial communities in each reactor.

Nitrospirota provides nitrite oxidation functional groups for the nitrification reaction (Pereira et al. 2014) and was the main NOB bacteria in the system, with a relatively high relative abundance in reactors (a) and (d), which was probably the reason for the faster nitrous nitrogen consumption in the above 0 and 30 mT reactors.

There were also relatively small amounts of phyla that functioned in the system: Actinobacteriota, Firmicutes, Myxococcota, and Elusimicrobia were the denitrifying bacteria in the reactor (Cai et al. 2020; Wang et al. 2022), and Chloroflexi was present as a potential NOB bacterium (Wang et al. 2022). Acidobacteriota has the potential for polyphosphate and glycogen accumulation, fermentation, and the reduction of nitrate to nitrite and ammonia (Kristensen et al. 2021).

In summary, different kinds of aerobic, anaerobic, and facultative microorganisms functioned in the SND system together and promoted the removal of nitrogen from the system. The relative abundance of these bacteria was higher in the magnetic reactor, which may have been one of the reasons for the advantages in the removal of total nitrogen in the magnetic reactor mentioned earlier.

In this study, MSND system was established and achieved efficient nitrogen removal in municipal sewage treatment. The optimized magnetic field intensity (20 mT) facilitated the short-range denitrification, and the carbon source balance in the denitrification process was achieved. The maximum nitrogen removal performance reached 89.60% in the magnetic field of 20 mT, and the maximum nitrogen removal load reached 0.18 kg N/(m³·day).

The magnetic fields could induce the succession of the AOB community structure, and the relative abundance of Proteobacteria and Bacteroidetes for nitrification and denitrification was enhanced. Alternatively, the abundance of related functional genes (i.e., gene proliferation and transmembrane transport) was increased, and thus the microbial activity and metabolic rate were promoted. In the future, the regulating mechanism by magnetic fields will be further explored using more monitoring analytical methods relating to spectroscopic and molecular biology, and the expanding test will be carried out. These results of this study have practical significance and are expected to provide a theoretical basis for municipal sewage treatment.

This work was financially supported by the National Natural Science Foundation of China (51808257).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.