Nitrogen removal performance of aerobic denitrifying bacteria enhanced by an iron-anode pulsed electric field

With the accelerated modernisation and rapid growth of the urban population, more nitrogenous compounds are being discharged into water bodies, causing various problems (Wang et al. 2022; Li et al. 2023; Xia et al. 2023). For example, excessive nitrates entering water bodies can cause eutrophication, acidification of water bodies, algal blooms, serious damage to local ecosystems and a sharp decrease in available water resources (Yan et al. 2022a, 2022b). Upon entering the body, nitrate converts to nitrite, which, in turn, converts the iron centre of haemoglobin from Fe²⁺ to Fe³⁺, yielding methaemoglobin, which is unable to bind and transport oxygen, ultimately causing methemoglobinemia (Brunato et al. 2003). Therefore, nitrogen pollution in water bodies is an important environmental issue that must be addressed urgently.

Currently, biological treatment is widely used for wastewater treatment because of its cost-effectiveness, high efficiency and low pollutant production (Zhang et al. 2020). Aerobic denitrification has been proposed as a novel technology for nitrate removal. The discovery of aerobic denitrifying bacteria disproved the conventional theory that denitrification can only occur under anaerobic or anoxic conditions (Ji et al. 2015). Aerobic denitrifying bacteria can use both oxygen and nitrate as electron acceptors for denitrification under aerobic conditions, facilitating the completion of the nitrification and denitrification processes in a single reactor, effectively reducing the treatment costs and operational complexity. Therefore, aerobic denitrification is widely used in various fields, such as wastewater treatment systems for domestic, industrial and agricultural wastewater. This technology is also widely used for the remediation of eutrophic water bodies, such as rivers and lakes, as well as for nitrate removal from groundwater (Yao et al. 2020).

To date, aerobic denitrification technology has been usually applied by directly injecting a certain amount of aerobic denitrifying bacteria into wastewater or activated sludge (Zhao et al. 2022). However, in the actual treatment process, the growth of aerobic denitrifying bacteria is restricted and their reproduction capacity decreases due to the constraints of growth conditions and competition among microbial populations, often rendering them incapable of becoming the dominant population (Bian et al. 2022). Meanwhile, in the treatment of high-nitrate-concentration wastewater, the denitrification process is inhibited to a certain extent with an increase in nitrite accumulation, which directly increases the growth time of aerobic denitrifying bacteria and decreases nitrate removal capacity; thus, meeting the long-term demand for high-nitrate-concentration wastewater treatment is difficult (Lan et al. 2022). Therefore, developing an efficient aerobic denitrification enhancement technology to fully use its advantages and potential is essential.

In this study, a PEF treatment system with iron as the electrode was developed to investigate the effect of PEF on the growth and metabolism of aerobic denitrifying bacteria. The objectives of this study were to (1) determine the optimal process parameters for stable operation of PEF–Fe treatment, (2) investigate the effect of PEF on the growth and metabolism of the aerobic denitrifying bacteria Pseudomonas putida W207-14 (W207-14) and (3) probe the cell membrane permeability of W207-14 via flow cytometry (FCM) combined with fluorescence staining analysis. The results of this study may provide new strategies for improving the denitrification capacity of PEF in wastewater treatment.

The aerobic denitrifying bacteria were isolated through laboratory screening and later identified as W207-14. The C/N ratio, initial pH and temperature of the artificially simulated wastewater (The artificially simulated wastewater contained (per litre): sodium succinate 7.5 g, KNO₃ 2.0 g, K₂HPO₄ 1.0 g, KH₂PO₄ 1.0 g, MgSO₄·7H₂O 0.2 g, 2.0 mL trace elements solution. All mediums were adjusted to pH 7.20 ± 0.01 and autoclaved for sterility at 121 °C for 20 min) were 15, 7.2 and 30 °C, respectively (suitable environment for W207-14 growth). All chemical reagents were of analytical grade. To investigate the effect of PEF on the growth and metabolism of W207-14, experiments were conducted at an initial pH of 7.2, temperature of 30 °C, rotational speed of 160 r·min⁻¹, different current intensities (0.01, 0.02, 0.03, 0.04 and 0.05 A), different pulse frequencies (100, 500, 1,000, 1,500 and 2,000 Hz), different electrode distances (2, 3, 4, 5 and 6 cm) and W207-14 inoculum of 5% using different iron ion dosing methods (S1 for W207-14, S2 for W207-14 + certain amount of iron ions, S3 for PEF and S4 for W207-14 + PEF). Moreover, optical density at 600-nm wavelength (OD 600) as well as chemical oxygen demand (COD) and –N removal rates, as the evaluation indexes, were used for these experiments. Subsequent experiments were conducted under optimal conditions.

The COD was determined via the hydrazine sulphate reduction using an ultraviolet–visible spectrophotometer (DR3900, Hashish Water Analysis Instruments Co., Ltd). Furthermore, –N was determined by the rapid extinction spectrophotometric method using an ultraviolet–visible spectrophotometer (Uvmini-1240, Shimadzu Corporate Management Co., Ltd). The sample morphology was observed using an ULTRA PLUS field-emission scanning electron microscope (SEM). FCM (FACSJazz) was conducted to observe the staining of the samples. Nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase were measured using ELISA Kits 48T. Moreover, Shanghai Bioengineering Co. was commissioned to conduct transcriptome sequencing of W207-14.

Statistical and preliminary analyses were performed using Microsoft Excel 2013. Descriptive statistical analyses were performed using IBM SPSS Statistics 22.0. Figures were created with Origin Pro 9.1 (32-bit). Three copies of each sample were taken for water quality analysis, with at least two biological copies for transcriptome analysis.

Under the low-frequency condition, the growth and metabolism of strain W207-14 were not inhibited and were at a high level, and its OD 600 value, COD and –N removal reached the maximum values of 1.724, 98.59% and 97.60%, respectively, at a pulse frequency of 500 Hz. Subsequently, the growth and metabolism of strain W207-14 were inhibited with the increase of pulse frequency to 2,000 Hz, and its OD 600 value, COD and –N removal rates showed a gradual decrease. As a large number of iron ions were released into the solution under the action of PEF–Fe, the phenomenon of agglomeration of some aerobic denitrifying bacteria was observed in the SEM, and at the same time, a small amount of flocs attached to the surface of some of the cells, which together formed a larger volume of ‘equivalent cells’. It has been confirmed that larger cells tend to be more tolerant to PEF, and under the condition that the intensity of PEF remains unchanged, a lower pulse frequency is often required to facilitate the accumulation of charge on the cell membrane, thus causing changes in the permeability of the cell membrane (Buchmann & Mathys 2019). Therefore, aerobic denitrifying bacteria subjected to PEF–Fe can withstand lower pulse frequency treatment without causing bacterial inactivation. With the increase of pulse frequency, it is unfavourable to the accumulation of charge on the cell membrane, which may lead to the reduction of the number of reversible electroporation holes on the cell membrane, thus affecting the nutrient uptake by the cells, so the growth and metabolism of strain W207-14 were inhibited (Gui et al. 2017). In summary, for aerobic denitrifying bacteria Pseudomonas putida W207-14, it is more appropriate to select a pulse frequency of 500 Hz for PEF–Fe treatment.

Compared with the aforementioned parameters, the electrode distance affected the growth and metabolism of W207-14 to a lesser extent (Figure 2(c)). The OD 600 value as well as the COD and –N removal rates of W207-14 increased and then decreased with increasing electrode distance. When the electrode distance was 5 cm, the OD 600 value and the COD and –N removal rates of W207-14 reached maximum values of 1.750, 98.35% and 96.51%, respectively. When the electrode distance was small, the number of W207-14 directly affected by PEF was small and their growth and metabolism were slow. With the increase in electrode distance, the electrostatic effect between the pole plates weakened and more iron ions were directly used by W207-14, which promoted their growth and metabolism. Subsequently, the growth and metabolism of W207-14 were inhibited when the electrode distance continued to increase to 6 cm. This may be because the mass transfer time of ions increases with an increase in electrode distance when the electrode distance exceeds the optimal distance, which hinders the PEF process (Daccache et al. 2020). In summary, an electrode distance of 5 cm was selected for subsequent experiments.

The OD 600 values and pollutant removal rates in the S4 treatment were higher than those in the other three treatments (Figure 2(d)). Compared with the S1 treatment, the S2 and S4 treatments provided better absorption and utilisation of iron ions by W207-14, while the S4 treatment enhanced the mass transfer efficiency in the system due to the stimulating effect of the applied electric field and stimulated the activity of W207-14, which could also be reflected through the significant differences in OD 600 values and pollutant removal rates. In the PEF treatment system, nitrate removal mainly occurred via electroflocculation and biological denitrification. In the S3 treatment, pollutant removal in the system was low for a short period of time, with COD and –N removal rates of only 12.37 and 18.03%, respectively, which may be due to the low current intensity and the difficulty for the electrode to directly denitrify H⁺ generated from the cathode as an electron donor in the absence of microbial catalysis (Daccache et al. 2020); therefore, degradation of pollutants via electroflocculation was considerably limited. The coupling of electroflocculation and biological denitrification in the S4 treatment afforded a significant increase in pollutant removal efficiency, with COD and –N removal rates of 98.93 and 97.60%, respectively. Compared with the OD 600 value of 0.439 in the S1 treatment, the OD 600 value in the S4 treatment was 1.741 for the same period of time, which confirmed that the removal of nitrate in PEF was mainly microbial, i.e. the iron ions precipitated by the iron electrode under the influence of the PEF were mainly absorbed by W207-14 for their growth and metabolism.

Aerobic denitrifying bacteria have heterotrophic nitrification, enabling nitrification and denitrification processes to be completed in one reactor at the same time, and ultimately realising the simultaneous removal of ammonia nitrogen and nitrate nitrogen (Bao et al. 2023). In the actual wastewater treatment process, the nitrification reaction product can directly act on denitrification to improve the system nitrogen removal efficiency, while simplifying part of the nitrogen removal process on the demand for different biological reactors, reducing sludge production and saving costs. In addition, the alkalinity generated in the denitrification process can partially compensate for the alkalinity consumption in the nitrification process, saving energy consumption. Therefore, aerobic denitrification technology has great economic potential and practical application value.

The effect of PEF on the nitrate reductase (Nap), nitrite reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase (Nos) activities of W207-14 was investigated; the results are shown in Table S1. Without PEF treatment, W207-14 exhibited Nap, Nir, Nor and Nos enzyme activities of 50.90, 33.41, 167.17 and 95.52 U·L⁻¹, respectively; after PEF treatment, W207-14 exhibited Nap, Nir, Nor and Nos enzyme activities of 73.81, 42.62, 229.68 and 132.71 U·L⁻¹, respectively. The Nap, Nir, Nor and Nos enzyme activities of W207-14 after PEF treatment increased by 45.01, 27.57, 37.39 and 38.93%, respectively. As confirmed in this study, PEF could improve the key enzyme activities of W207-14. Enzymes play a vital role in the life activities of living organisms, and they must be involved in the absorption of nutrients from the environment by microorganisms as well as their utilisation in the body. In the process of biological treatment, the decomposition and transformation of pollutants in wastewater by microorganisms are catalysed by enzymes in a series of complex biochemical reactions (Veneziani et al. 2019). Some studies have confirmed that iron ions are an important component of the aerobic respiratory electron transport chain (Simon & Klotz 2013). Additionally, iron ions are common inorganic cationic catalysts for enhancing enzyme activity and are often used as coenzyme catalysts. Furthermore, the denitrification process involves several proteins that require metal ions as cofactors, among which iron ions are required for the cytochrome subunits of nitrate reductase and nitrite reductase. The results of this experiment also confirmed that the PEF action had a significant effect on the key enzyme activity of strain W207-14, which explains, from an enzymatic point of view, that the increase in the activity of the denitrification key enzyme of strain W207-14 after treatment with PEF–C is one of the key reasons for the significant increase in the rate of nitrate removal.

PI is a membrane-impermeable dye that cannot enter cells with intact membranes; however, when cell membranes are damaged or perforated, PI can enter cells and bind to nucleic acids showing red fluorescence (Davey & Hexley 2011) (Figure 5(a) and 5(b)). Almost all cells without PEC treatment before PI staining were located in the Q4 quadrant and the percentage was ∼98.3%, indicating that W207-14 without PEF treatment had intact cell membranes and did not exhibit cell membrane breakage or perforation. The majority of the cells after PEF treatment moved to the Q1 quadrant and the proportion of cells in the Q1 quadrant increased to 82.2%, with 17.8% of cells still located in the Q4 quadrant, indicating that the cell membrane of W207-14 after PEF treatment underwent electroporation, which led to PI entering through the damaged cell membrane and binding to nucleic acids. The proportion of cells showing red fluorescence via PI staining significantly increased; thus, most cells moved from the Q4 quadrant to the Q1 quadrant. Therefore, PEF induced the change in the cell membrane permeability of W207-14.

Because the survival status of cells that can be stained by PI is still controversial, this experiment introduced another staining reagent, CFDA SE, to confirm the physiological status of the cells (Zhou et al. 2020). The hydrolysis product Crystal Field Splitting Energy, (CFSE) accumulates and appears fluorescent only in cells with esterase activity, whereas dead cells are not stained (Zhao et al. 2011) (Figure 5(c) and 5(d)). The majority of the cells without PEF treatment before CFDA SE staining were located in the Q3 quadrant, with a proportion of ∼88.3%, indicating that most W207-14 cells had high esterase activity and could be identified as viable cells and only a small proportion of W207-14 was unstained or lost esterase activity. After PEF treatment, 75.4% of the cells were still located in the Q3 quadrant, which was slightly lower than the percentage of the untreated cells. The results indicate that PEF did not cause widespread inactivation of W207-14 and most W207-14 cells were able to survive and maintain high physiological activity in the presence of PEF.

Because the staining information obtained via PI or CFDA SE single staining is relatively single, FCM combined with PI–CFDA SE double staining can more visually observe the changes in cell membrane permeability and esterase activity of the aerobic denitrifying bacteria treated with PEF at the same time (Figure 5(e) and 5(f)). The majority of the cells without PEF treatment before PI–CFDA SE double staining were located in the Q3 quadrant, with a proportion of ∼83.8%, indicating that most W207-14 cells without PEF treatment had high esterase activity while the cell membrane was intact. The majority of the cells treated with PEF moved towards the Q2 quadrant and the proportion of cells in the Q2 quadrant increased to 71.6%, with another 25% moving to the Q1 quadrant, indicating that the PEF-induced cell membrane damage did not cause widespread inactivation of W207-14. Moreover, this result indicated that most of the W207-14 cells maintained high physiological activity despite changes in cell membrane permeability and PEF was similarly able to induce reversible electroporation on the cell membrane. We hypothesised that the damage to the cell membrane caused via PEF-induced reversible electroporation is recoverable sublethal damage. The mechanism of microbial inactivation via PEF is based on the leakage of components caused via irreversible electroporation on the cell membrane. Moreover, during recovery of reversible electroporation at low electric field intensity, the improvement in cell membrane permeability promoted the uptake of nutrients in the medium by microorganisms (Johnson et al. 2010).

The changes in the transcriptome of W207-14 before and after PEF treatment were explored at the same growth time (8 h) during the growth stabilisation period, where sample A represents W207-14 without PEF treatment and sample D represents W207-14 with PEF treatment. Principal component analysis (Fig. S1) showed that the biological replicates within the respective groups of samples A and D were close to each other with high reproducibility, reflecting the reliability of the transcriptome sequencing test results (Xu et al. 2022). Meanwhile, the distances between the samples under different treatment conditions between the groups of samples A and D were large and there was a large variability, indicating that PEF-induced substantial transcriptional changes in W207-14.

All differentially expressed genes of W207-14 before and after PEF treatment were classified into three major functional categories: biological processes, cellular components and molecular functions (Fig. S4). Among them, in the biological processes category, differentially expressed genes are commonly found in several GO Terms such as cellular process (GO:0009987), metabolic process (GO:0008152), biological regulation (GO:0065007), stress response (GO:0050896) and biological process regulation (GO:0050789). In the cellular components category, differentially expressed genes are commonly found in several GO Terms such as cell (GO:0005623), cellular fraction (GO:0044464), cell membrane (GO:0016020), cell membrane fraction (GO:0044425) and protein complex (GO:0032991). Furthermore, in the molecular function category, differentially expressed genes are commonly found in several GO Terms such as catalytic activity (GO:0003824), binding (GO:0005488) and transport activity (GO:0005215).

The results indicate that PEF induced different levels of stress responses in the growth and metabolism of W207-14. The PEF-treated W207-14 maintained normal life activities through the regulation of genes involved in cellular and metabolic processes and other related genes to adapt to the changes in the growth environment created via PEF. Moreover, to obtain sufficient energy for growth and metabolism under the influence of PEF, W207-14 needs to enhance its uptake of nutrients and its ability to bind to different ions by regulating the expression of genes related to catalytic activity, binding and electron transfer (Chueca et al. 2015). The abundant changes in the expression of genes related to the cell membrane in the cell fraction category confirm that the cell membrane is one of the main targets of PEF action on microorganisms.

The results showed that PEF–Fe action could influence the metabolic process of strain W207-14. The highest percentage of GO Term related to metabolism was found among the top 30 significantly differentially expressed genes GO Term. It was confirmed that strain W207-14 must regulate the expression of metabolism-related genes in different degrees to maximise the amount of energy from organic matter for growth and metabolism in a stressful environment. At the same time, cell membrane repair must be completed to adapt to the stimulation from the PEF–Fe environment and maintain normal life activities without being inactivated. The effect of PEF–Fe can affect the aerobic respiration of strain W207-14. Aerobic denitrifying bacteria use the same fundamental mechanism of aerobic respiration via the aerobic respiratory electron transport system (respiratory chain) for both aerobic respiration and denitrification processes (Yang et al. 2020). The enhanced expression of these genes located near aerobic cellular respiration helps facilitate electron transfer during aerobic respiration when subjected to PEF–Fe treatment, providing sufficient energy for the vital functions of strain W207-14 (Hao et al. 2022). The use of PEF–Fe can influence the redox process of strain W207-14, which is a crucial aspect of conventional biological denitrification. In this process, NH₄⁺-N is oxidised to -N and gradually reduced to N₂ under aerobic conditions. The nitrogen metabolism process of strain W207-14 can be performed smoothly under a PEF–Fe stress environment by differentially regulating the expression of redox-related genes under the action of PEF–Fe, which can affect the iron–sulphur cluster binding of strain W207-14. The iron–sulphur cluster is a fundamental redox centre that has been present in all living organisms for a long time and plays a critical role in cellular metabolism (Braymer et al. 2021). Aerobic denitrifying bacteria often use periplasmic nitrate reductase (Nap) containing an [4Fe–4S] iron–sulphur cluster as a marker for aerobic denitrification (Pandey et al. 2020). Strain W207-14 can regulate the expression of genes related to the iron–sulphur cluster and redox enzymes in conjunction with PEF–Fe treatment, enabling aerobic denitrification. The action of PEF–Fe improves the efficiency of the aerobic denitrification process.

Figure S4 and Table S2 show directed acyclic plots of the significant GO Term under the functional classification of cellular fraction GO for the PEF–Fe-treated strain W207-14, which was selected for enrichment correlation analysis in conjunction with the previous effect of PEF–Fe on strain W207-14.

Under the functional classification of cellular components, the GO Term branch of the cell membrane (GO: 0016020) has a significantly differentially expressed gene, the membrane protein complex (GO: 0098796). Among the 20 upregulated genes in the membrane protein complex GO Term, 10 encode ABC transporter proteins. These proteins are widely found in various organisms and can use Adenosine triphosphate,(ATP); hydrolysis-generated energy to transport organic substances bound to them across membranes (Wu et al. 2023). The ABC transporter proteins can transport various substances across membranes using two mechanisms: inward and outward transport. Based on the transport mechanism of these proteins, it is hypothesised that under the action of PEF–Fe, reversible electroporation occurs on the cell membrane surface of aerobic denitrifying bacteria. This improves the cell membrane permeability, allowing the transporter protein to facilitate the inward transport of nutrients such as sugars and amino acids from the extracellular environment to the intracellular matrix (Elbourne et al. 2019). This promotes cell growth and metabolism. Additionally, the outward transport process can also aid in ‘detoxification’ by transferring substances that are harmful to the growth of W207-14 cells out of the cells. This helps to keep the non-essential exogenous substances or secondary metabolites at a low concentration within the cells, reducing growth pressure and maintaining normal cell growth, which greatly improves the survival rate of the cells.

Studies have identified the following three strategies for regulating cell membrane permeability: (a) lipid-mediated membrane permeability regulation; (b) regulation of membrane protein function; and (c) regulation of the intracellular membrane energy system (Choudhuri & Klaassen 2018). The ABC transporter protein is a membrane channel protein that regulates the entry and exit of substances into and out of the membrane using the energy generated by ATP hydrolysis in the cytoplasm. This protein can influence both the function of membrane protein and the intracellular membrane energy system. PEF–Fe treatment activates the transmembrane transport function of ABC transporter protein and enhances cell membrane permeability by regulating membrane protein function and the intracellular membrane energy system. This ultimately leads to a significant increase in the growth rate and nitrate removal rate of strain W207-14, providing a molecular biological explanation for the previous findings.

Figure S5 shows that significant GO Term directed acyclic plots of strain W207-14 after PEF–Fe treatment under molecular functional GO functional classification. Table S3 presents the significant differentially expressed genes. Iron–sulphur clusters play an important role in the intracellular energy regulation of W207-14 in the PEF–Fe system. The primary function of the iron–sulphur cluster is electron transfer, which is involved in intracellular energy transfer as a cofactor group of electron transfer proteins (Pauleta et al. 2023). The iron–sulphur cluster uses electrons generated by PEF–Fe interaction to convert iron ions from the oxidised state to the reduced state, followed by the transfer of electrons to other receptor proteins and the return to the initial oxidised state, thus starting a new electron transfer. Through the energy regulation of the iron–sulphur cluster, the strain W207-14 within the PEF–Fe system is always in a suitable energy state. The iron–sulphur cluster has a certain catalytic effect and can participate in the system's binding and activation of substrates (Boncella et al. 2022). For instance, periplasmic nitrate reductase (Nap), a hallmark of aerobic denitrification and an iron–sulfur cluster in the form of [4Fe–4S] in Nap could better facilitate the process of aerobic denitrification (Flamholz & Newman 2022). The PEF–Fe system precipitates many Fe ions, and the iron–sulphur cluster can participate in intracellular iron storage and maintain the dynamic balance of intracellular iron ions so that the growth metabolism of strain W207-14 can better use iron ions. Compared with the control group, a further significant increase in the growth rate and nitrate removal rate of strain W207-14 under the action of PEF–Fe was inextricably linked to the regulation of iron–sulphur clusters in the aerobic denitrification process.

The growth and metabolism of W207-14 were enhanced through PEF treatment. The growth time required for W207-14 to achieve the maximum OD 600 value was reduced by five times and the nitrate removal rate increased by 599.14% after PEF treatment compared with those of untreated W207-14. Using SEM, a smaller size was observed after PEF treatment. Moreover, FCM combined with fluorescence staining analysis revealed that the PEF-treated cells maintained higher physiological activity while the cell membrane was damaged, confirming the reversible electroporation of the membrane surface of the PEF-treated W207-14 cells. Differentially expressed gene enrichment analysis indicated that PEF–Fe treatment activated the transmembrane transport function of the ABC transport proteins, which led to enhanced cell membrane permeability in aerobic denitrifying bacteria. Additionally, the significant differential expression of iron–sulphur cluster proteins facilitated the regulation of electron transport and maintenance of the dynamic balance of iron ions within the PEF–Fe system.

The research was supported by the Liaoning Provincial Science and Technology Plan Project ‘Application Research on Solidification and Resourcing of Domestic Waste Incineration Fly Ash Based on Geopolymerization Theory’ (2020JH2/10300094) and the Liaoning Provincial Central Leading Local Science and Technology Development Special Project ‘Application Research on Solidification and Resourcing of Waste Incineration Fly Ash and Waste Concentrate’ (2021JH6/10500217). Special thanks are due to the instrumental or data analysis from Analytical and Testing Center, Northeastern University, China and Shiyanjia Lab (www.shiyanjia.com).

F. W. investigated the article, experimented he data, conceptualised the whole article, conducted formal analysis, and rendered support in data curation; B. Z. conducted formal analysis, wrote the original draft, wrote the review and edited the article; X. D. reviewed the article; X. Z. reviewed the article; X. H. reviewed and supervised the article, administered the project.

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

The authors declare there is no conflict.