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Systematic Review

Optimizing the Nitrogen Removal Efficiency of an Intermittent Biological Sponge Iron Reactor by Immobilizing Aerobic Denitrifying Bacteria in the Biological Sponge Iron System

by
Jing Li
1,
Jie Li
1,*,
Yae Wang
1,
Hao Mu
2,
Huina Xie
1 and
Wei Zhao
1
1
College of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
2
College of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710000, China
*
Author to whom correspondence should be addressed.
Water 2025, 17(9), 1308; https://doi.org/10.3390/w17091308
Submission received: 27 March 2025 / Revised: 21 April 2025 / Accepted: 26 April 2025 / Published: 27 April 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

:
This study investigates the enhancement of nitrogen removal performance in an intermittent biological sponge iron system (BSIS) through the immobilization of aerobic denitrifying bacteria. The aim is to improve the efficiency of simultaneous nitrification and denitrification (SND) in the BSIS by optimizing the microbial community involved in nitrogen conversion. The immobilization technique not only stabilizes the microbial activity and abundance of aerobic denitrifying bacteria, but also promotes a more efficient denitrification process. The optimal material ratio of polyvinyl alcohol–sodium alginate gel beads was determined as 10 g/100 mL PVA, 4 g/100 mL SA, 2 g/100 mL CaCl2, and 2 g/100 mL of bacterial suspension, achieving a maximum NO3−-N removal rate of 91.73%. A response surface model (RSM), established for the operational conditions, (shaker speed, temperature, and pH) showed a high fitting degree (R2 = 0.9960) and predicted the optimal conditions for maximum NO3−-N removal as 109.24 rpm, 23.6 °C, and pH 7.9. Compared to R1 (47.82%), R3 achieved a higher average total nitrogen (TN) removal rate of 95.49%, following the addition of immobilized aerobic denitrifying bacteria to the BSIS.

Graphical Abstract

1. Introduction

Nitrogen is an essential element for living organisms, but the excessive release of organic nitrogen and inorganic nitrogen into water bodies can cause serious environmental problems [1], such as the deterioration of air quality and water eutrophication. These issues not only disrupt the ecological balance, but also pose serious risks to human health and the growth of animals and plants [2]. Nitrates are generally non-toxic at low concentrations, but when human intake exceeds a certain threshold, they can cause various diseases, including blue baby syndrome and methemoglobinemia [3], posing a threat to health [4]. Nitrites can oxidize the iron in hemoglobin from Fe2+ to Fe3+, forming methemoglobin, which then hinders the normal transport of oxygen. Additionally, nitrites may react with tertiary amines to form carcinogenic nitrosamines in infants and ruminants, leading to various physiological disorders and immune suppression, or causing severe diseases in aquatic animals [5]. Ammonia nitrogen not only promotes eutrophication of surface water, but can also causes respiratory diseases [6]. Traditional wastewater nitrogen removal processes primarily rely on autotrophic nitrification and anaerobic denitrification as biological nitrogen removal technologies. However, factors such as toxic substance shocks [7], low temperatures [8], and others, may lead to system instability and performance deterioration.
Enhancing the efficiency of aerobic denitrification is key to optimizing the nitrogen removal effect during wastewater treatment processes. Chen et al. [9] investigated the application of Acinetobacter in a pig farm wastewater treatment plant in regard to nitrogen removal and found that it effectively removed nitrate at a rate of 553 mg L−1 h−1. Huang et al. [10] used heterotrophic and autotrophic denitrification methods to remediate groundwater contaminated with nitrate. Guo et al. [11] applied Pseudomonas to perform simultaneous nitrification–aerobic denitrification in eutrophic lake water. Zheng et al. [12] used psychrophilic bacteria to treat water in marine aquaculture systems, achieving 100% nitrate removal. Liu et al. [13] found that Corynebacterium could be used for bioaugmentation to remove nitrogen (NH4+-N and NO3−-N) using a moving bed biofilm reactor, operating under aerobic conditions. He et al. [14] utilized an aerobic granular sequencing batch reactor for simultaneous nitrification–aerobic denitrification. Ji et al. [15] inoculated Ps stutzeri into an aerobic upflow biofilter and achieved a nitrate removal rate of 98.48%, with an initial nitrate concentration of 63.0–73.8 mg/L. In recent years, some studies have explored the bioaugmentation effects of aerobic denitrifying bacteria in reactors [16]; Tang et al. [17] studied the feasibility of using biostimulants to bioaugment urban river sediments to improve the nitrogen removal efficiency of aerobic denitrifying bacteria and reported that the combined technology of bioaugmentation and biostimulation increased the removal rate of nitrate (NO3−-N). However, due to its poor competitive ability in the environment compared to dominant microbial communities, it is difficult to form a dominant group of aerobic denitrifying bacteria, and, thus, long-term effective nitrogen removal enhancement cannot be achieved.
The biological sponge iron system (BSIS) is a novel enhanced biological nitrogen removal technology, which involves incorporating sponge iron (SFe) into the activated sludge system in a certain manner to form a mixed system [18]. It is a fusion of iron–carbon microelectrolysis (IC-ME) and the traditional activated sludge method [19]. SFe corrodes and releases Fe2+ ions, and numerous studies have shown that: (1) Fe2+ not only promotes the synthesis of microbial nitrogen removal-related enzymes, but also further stimulates the activity of the microorganisms themselves [20]; and (2) as the active center of NADH dehydrogenase, ubiquinone, cytochrome c, and cytochrome bc1, the interconversion of Fe2+ and Fe3+ is of great significance in the cellular electron transport chain [21]. In addition, due to the special structure of SFe [22], the BSIS forms local anaerobic and anoxic microenvironments, thereby increasing the relative abundance of aerobic denitrifying bacteria [23]. Li et al. [24] found that the TN removal rate of the BSIS, coupled with a polyurethane foam system, was 5.2% higher compared to the activated sludge system coupled with a polyurethane foam system, which is attributed to the BSIS having a certain simultaneous nitrification and denitrification (SND) capability. The involvement of SFe can significantly alter the original microbial community structure of the activated sludge system, resulting in a large number of nitrogen removal-related iron bacteria being produced in the BSIS. Simultaneously, the aerobic denitrifying bacteria, Aquabacterium [24], Acidovorax [25], and Rhodanobacter [26], with an iron oxidation function, were found in the BSIS, which further explains why the BSIS possesses simultaneous nitrification and denitrification (SND) capability.
Immobilized microbial technology is based on a bioaugmentation technique that involves fixing free microorganisms onto a specific carrier surface or within it through physical or chemical methods, while maintaining microbial activity [27]. It can not only effectively improve the microorganisms’ resistance to load and shock [28], but also achieve stable microbial proliferation and prevent the loss of dominant strains [29,30]. Embedding is the most studied microbial immobilization technique, which involves encapsulating dominant microorganisms or strains within semi-permeable porous polymers or gels, allowing small molecule substrates and metabolic products to freely pass through, without the microorganisms leaking out. This method has advantages such as simple operation, minimal loss of microbial activity, high microbial capacity, high mechanical strength of the immobilized particles [31], and good chemical stability [32]. Common embedding methods include gel embedding, microencapsulation, emulsified liquid membrane embedding, and preformed material embedding [33,34]. Commonly used embedding agents include polyvinyl alcohol (PVA), sodium alginate (SA), carrageenan, agar, and polyacrylamide (PAM) [33,35,36]. Sodium alginate (SA) is a natural organic polymer material [37] that is non-toxic to microorganisms and has high mass transfer efficiency [38], but the immobilized gel beads produced have poor bead formation and low mechanical strength [39]. Polyvinyl alcohol (PVA) as a new type of immobilization carrier has high mechanical strength and good chemical stability, is non-toxic to microorganisms, and has strong resistance to microbial decomposition [40], but its mass transfer performance is poor. Preparing PVA–SA gel beads can effectively improve the mechanical strength [41], chemical stability [42], and biocompatibility of the gel beads [43,44], and enhance their ability to operate stably in complex wastewater treatment systems [35]. Yu et al. [45] used PVA–SA material to immobilize shortcut nitrification sludge, achieving a nitrite accumulation rate (NAR) of 99% within 140 h, at a DO concentration of 0.5~2 mg/L. Bae et al. [46] used PVA–SA to immobilize AOB and Anammox sludge, achieving a nitrogen removal efficiency of 80.4% ± 1.20% and maintaining a stable period of 30 days. Liu et al. [47] used PVA–SA to immobilize traditional nitrification sludge and inhibited NOB with 10 g/L of NaCl, maintaining an NAR of over 70% for 30 days.
In this study, polyvinyl alcohol–sodium alginate (PVA–SA) was used to immobilize multiple strains of aerobic denitrifying bacteria isolated from the intermittent biological sponge iron system. Orthogonal tests were employed to optimize the best ratio of the immobilization material, and the response surface methodology was used to optimize the operating conditions of the immobilized gel beads. This study aims to investigate the bioaugmentation effect of aerobic denitrifying bacteria and their immobilization on the simultaneous nitrification and denitrification (SND) capability of the BSIS, achieving stable and efficient SND in the SBR reactor and providing parameter references and experimental support for the practical application of the BSIS in the future.

2. Materials and Methods

2.1. Physicochemical Properties of the Experimental Wastewater

To ensure the stability of the water concentration for the experiment, the water used in this experiment was simulated domestic sewage, to which 2 mL of trace element stock solution was added per liter of tap water. The physicochemical properties of the simulated domestic sewage are as follows: 191.1 mg/L of NH4Cl, 96 mg/L of NaHCO3, 384.9 mg/L of CO3COONa, 29.2 mg/L of K2HPO4, and 11.5 mg/L of KH2PO4. The trace element stock solution’s physicochemical properties are as follows: 50 g/L of EDTA, 7.28 g/L of CaCl2·2H2O, 5 g/L of FeSO4·7H2O, 3.92 g/L of ZnSO4·7H2O, 2.06 g/L of MnCl2·4H2O, 1.1 g/L of (NH4)6Mo7O24·4H2O, 1.57 g/L of CuSO4·5H2O, and 1.61 g/L of CoCl2·6H2O.

2.2. Isolation and Culturing Aerobic Denitrifying Bacteria

The activated sludge sample used for the isolation of aerobic denitrifying bacteria was sourced from a well-operated BSIS managed by the research group. The sample (10 mL) was inoculated in a 250 mL conical flask and cultured at 30 °C and 160 rpm for 30 min, after which it was diluted with sterile water, using the gradient method. The diluted solution (0.5 mL) was evenly inoculated onto a BTB medium (Table S1), coated using a swab stick, and cultured in an incubator at a constant temperature of 30 °C. The BTB medium was supplemented with the acid-base indicator, bromothymol blue, which changes from green to blue when the pH increases from 7 to higher levels. If the bacteria have denitrification capabilities, the pH of the medium around the bacterial colonies will increase, causing the color of the colonies to turn blue [48]. After the appearance of a blue halo on the medium, individual colonies with a larger halo and deeper color were selected for isolation and purification, using the streak plate method. The isolated strains were inoculated onto medium at 24 h, 30 °C, and 160 rpm, and then transferred to a 250 mL conical flask containing 100 mL of DM, sealed with 8 layers of gauze, and cultivated for 48 h. After cultivation, the aerobic denitrification performance of the bacteria was measured at 30 °C and 160 rpm.
After cultivation, the contents of NO3−-N, NO2−-N, and NH4+-N in DM were measured, and each strain was subjected to three parallel experiments to screen for aerobic denitrifying strains with high nitrogen removal potential for culture enrichment. Using a sterile sampling tube, 10 mL of aerobic denitrifying bacterial solution was taken, allowed to settle for 5 min, and then the supernatant was discarded. Sterile water was added back to achieve a volume of 10 mL, and this process was repeated twice. Subsequently, the bacterial solution was placed in a centrifuge and centrifuged at 4000 rpm for 10 min. After centrifugation, the supernatant was discarded, and the volume was brought back to 10 mL using sterilized normal saline to prepare the aerobic denitrifying bacterial suspension.

2.3. Preparation of Immobilized Aerobic Denitrification Gel Beads

Polyvinyl alcohol and sodium alginate were dissolved in distilled water at 100 °C, and the resulting mixture was cooled to room temperature. Then, using a sterilized pipette, 10 mL of the previously prepared aerobic denitrifying bacterial suspension was added to the mixture. Prepare a hot saturated boric acid solution by adding an excess amount of boric acid to hot water and stirring thoroughly. Allow the solution to cool and then collect the supernatant for later use. Weigh an appropriate amount of CaCl2 and dissolve it in the saturated boric acid solution to prepare a 1% CaCl2 saturated boric acid solution. Adjust the pH of the solution to 6.7 ± 0.1 using NaHCO3. The polyvinyl alcohol–sodium alginate–aerobic denitrification bacterial mixture was slowly dropped into the saturated boric acid–CaCl2 solution from a height of 20 cm, using a 10 mL sterile syringe, while a glass rod was used to slowly stir the solution to prevent the beads from sticking together. After that, the beads were left to crosslink in a 4 °C refrigerator for 24 h. Once crosslinking was complete, the beads were rinsed three times with saline solution and then stored in a refrigerator at 4 °C for later use.

2.4. Optimization of Immobilized Material Formulation

To study the effects of different factors on the degradation of NO3−-N by immobilized aerobic denitrifying bacteria, batch experiments with different concentrations were conducted: SA-3 g/100 mL, 4 g/100 mL, and 5 g/100 mL; PVA-9 g/100 mL, 10 g/100 mL, and 11 g/100 mL; and CaCl2-2 g/100 mL, 3 g/100 mL, and 4 g/100 mL. The bacterial community embedding amounts were as follows: 1 g/100 mL, 2 g/100 mL, and 3 g/100 mL. A four-factor three-level orthogonal test was designed to optimize the proportion of each component material used in the preparation process, and the optimal conditions obtained from the test were as follows: a PVA of 10 g/100 mL, and SA of 4 g/100 mL, and CaCl2 of 2 g/100 mL. The bacterial community embedding amount was 2 g/100 mL. Subsequently, using the Box–Behnken design of the response surface method, the effects of the shaker speed, temperature, and pH on the NO3−-N removal performance of immobilized aerobic denitrifying bacteria gel beads were investigated.

2.5. Evaluation of the Denitrification Capacity of Immobilized Aerobic Denitrifying Bacteria

R1 (BSIS), R2 (BSIS + free-state aerobic denitrifying bacteria), and R3 (BSIS + immobilized aerobic denitrifying bacteria) were initiated in conical flasks, with an effective volume of 200 mL. The control group, R1, consisted of conventional activated sludge, with a mixed liquid suspended solids (MLSSs) concentration of 1920 mg/L. SFe (with a particle size of 3–5 mm and a dosage of 90 g/L) was filled into polypropylene biochemical suspension beads, with a diameter of 4 cm, and were evenly distributed in the reactor in a suspended manner, forming the BSIS. Ten percent of aerobic denitrifying bacteria and 10% of immobilized aerobic denitrifying bacteria gel beads were inoculated into two reactors based on RI, forming R2 and R3. This study investigated the denitrification performance of the BSIS after the introduction of immobilized aerobic denitrifying bacteria. Each reactor was operated continuously involving a sequence of influent (15 min), aeration (11 h), sedimentation (30 min), effluent (15 min), per cycle, with a water replacement ratio of 1/2, accumulating a total of 30 days of operation.

2.6. Statistical Analysis

The COD, NH4+-N, NO3−-N, NO2−-N, and TN were measured (Table S2), according to the specific standard method previously described (Ahmad et al.). The data were plotted using Origin 2025. All the experiments were repeated three times, and the data are expressed as the mean ± standard deviation.

3. Results

3.1. Isolation of Aerobic Denitrifying Bacterial Communities

Through enrichment, BTB (bromothymol blue) plate streaking, and the applied purification steps, 45 bacterial strains with aerobic denitrification capability were successfully isolated from the BSIS, as shown in Figure 1. Subsequently, the aerobic denitrification capacity of each strain was tested. Finally, the top three strains (L-11, L-33, L-41) with the best aerobic denitrification ability were selected for subsequent experiments. Based on this identification, strain L-11 was identified as Achromobacter denitrificans, strain L-33 as Delftia lacustris (β-Proteobacteria, accession number ON159555), and strain L-41 as Pseudomonas putida (γ-Proteobacteria).

3.2. Immobilization Material Ratio Optimization

Using polyvinyl alcohol (PVA) and sodium alginate (SA) as embedding agents, and CaCl2 as a crosslinking agent, immobilized aerobic denitrifying bacterial gel beads were prepared (Table S3). Through the use of single-factor experiments, with the ball-forming performance of the gel beads as the indicator, a set of orthogonal experiments with four factors and three levels was designed, based on the conditions of SA (4 g/100 mL), PVA (10 g/100 mL), and CaCl2 (3 g/100 mL), and the amount of the bacterial community embedded (2 g/100 mL). The removal rate of NO3−-N by the gel beads was used as the evaluation index for nine groups of experiments, to optimize the ratio of each component material in the preparation process for immobilized aerobic denitrifying bacterial gel beads. Meanwhile, range analysis was used to determine the component material that had the greatest impact on the aerobic denitrification performance of the gel beads during the preparation process.
The final results of the orthogonal tests are presented in Table 1. K1, K2, and K3 represent the total sum of response indices for each factor at different levels, while k1, k2, and k3 are the weighted average values of K1, K2, and K3, respectively. The range R indicates the maximum difference in the weighted average values of each factor at different levels, which is used to measure the significance of each influencing factor on the response index. A larger R value indicates a more significant impact.
The analysis results show that the optimal dosage of SA is at level 2, which is 4 g/100 mL, with a corresponding R value of 2.45; the optimal dosage of PVA is at level 2, which is 10 g/100 mL, with a corresponding R value of 5.36; the optimal dosage of CaCl2 is at level 1, which is 2 g/100 mL, with a corresponding R value of 1.30; and the optimal level for the embedding amount of aerobic denitrifying bacteria is at level 2, which is 2 g/100 mL, with a corresponding R value of 4.87. Through the use of range analysis, the order of influence of the four factors on the NO3−-N removal rate was found to be as follows: PVA dosage > bacterial community embedding amount > SA dosage > CaCl2 dosage.
Under the optimal material ratio conditions (PVA: 10 g/100 mL, SA: 4 g/100 mL, CaCl2: 2 g/100 mL, bacterial embedding amount: 2 g/100 mL), the immobilized gel beads achieved the highest NO3−-N removal rate of 91.73%. These findings resulted in the adoption of the optimal ratio, as determined by the orthogonal experiment, for all subsequent experiments.

3.3. Response Surface Methodology Experimental Results

3.3.1. Model Establishment and Predictive Optimization

Five 250 mL conical flasks were charged with varying masses of immobilized gel beads: 5 g, 10 g, 15 g, 20 g, and 25 g. The treatment experiments were conducted over a series of 10 cycles using a shaker platform, with a constant agitation rate of 150 revolutions per minute (rpm) and a controlled operational temperature of 30 °C. Throughout the experimental period, the total nitrogen (TN) concentration in the effluent from each of the five reactors was quantitatively monitored. The findings indicated that the optimal TN removal efficiency and biological denitrification performance were attained when 10 g of gel beads were employed in the treatment involving 100 mL of simulated nitrate–nitrogen (NO3−-N) wastewater. Subsequently, with the optimal gel bead dosage determined, further experimentation was conducted.
Based on the Box–Behnken design, 17 groups of experiments were conducted in 250 mL conical flasks, with the addition of 10 g immobilized aerobic denitrifying bacterial gel beads. After four cycles of pretreatment operation, the corresponding indicators were measured. The response surface experimental results are shown in Table 2.
Establish a quadratic model relating the removal rate of NO3−-N in gel beads to the three environmental factors (shaking speed, temperature, and pH):
Y = − 60.44325 + 1.20373 × X1 + 3.22265 × X2 + 12.74725 × X3 − 0.00033 × X1X2 − 0.005775 × X1X3 − 0.014375 × X2X3 − 0.005081 × X12 − 0.073085 × X22 − 0.8065 × X32
In the formula, Y represents the NO3−-N removal rate (%) of the gel beads, and X1, X2, and X3 are the coded values for the shaking speed, temperature, and pH, respectively.
Table 3 and Figure 2 shows the analysis of variance (ANOVA) results for the quadratic model. The results indicate that the p-values for the shaking speed, temperature, and pH are all less than 0.0002, with an R2 = 0.9960 and a signal-to-noise ratio = 40.9559. This suggests that the quadratic model has a good fit and can be used for the analysis, prediction, and optimization of the NO3−-N removal rate of the gel beads.

3.3.2. Residual Analysis

The residual distribution plot of the fitted model is shown in Figure 3. The residuals of the model fluctuate near the fitting line, and the predicted values produced by the model basically match the actual values, indicating that the quadratic model has a high degree of fit and can be used for prediction and evaluation purposes.
The importance of environmental factors on the NO3−-N removal rate of the gel beads is as follows: shaking speed > temperature > pH. This result is similar to the conclusions drawn in other studies [49,50]. Based on the model, the influencing factor conditions were optimized using Design Expert 13. The predicted results indicate that the optimal process conditions for denitrification by immobilized aerobic denitrifying bacteria gel beads are a shaking speed = 109.24 rpm, a temperature = 23.6 °C, a pH = 7.9, with a predicted maximum NO3−-N removal rate of 89.22%. Under these conditions (110 rpm, 23.6 °C, pH 7.9), three parallel experiments were conducted, and the NO3--N removal rate reached 90.58% during the validation experiments, indicating that the model fits well with the experimental data.

3.3.3. Analysis of Response Surface Plots and Contour Plots Among Various Environmental Factors

The interaction relationships between the shaking speed and temperature, the shaking speed and pH, and the temperature and pH are shown in Figure 4. The results indicate that the interaction between the shaking speed and temperature is not significant (0.7386). Moreover, the change in shaking speed has a greater impact on the NO3−-N removal efficiency of the immobilized aerobic denitrifying bacteria gel beads than the change in temperature. The change in shaking speed has a greater influence on the NO3−-N removal efficiency than the change in pH, and the change in temperature has a greater impact on the NO3−-N removal efficiency than the change in pH. The interaction among the three environmental factors is not significant. This is different from previous reports, which stated that the TN removal rate of Corynebacterium pollutisoli SPH6 was significantly affected by the interaction between pH and C/N [13].
Therefore, the shaking speed is the decisive factor affecting the NO3−-N removal efficiency of immobilized aerobic denitrifying bacteria gel beads prepared using the optimal ratio (PVA: 10 g/100 mL, SA: 4 g/100 mL, CaCl2: 2 g/100 mL, bacterial embedding amount: 2 g/100 mL). When the shaking speed is too low, the gel beads’ NO3−-N removal efficiency significantly decreases, which may be due to the insufficient shaking speed leading to a low dissolved oxygen concentration within the reactor. The gel beads have a certain resistance to oxygen transmission, which prevents the key enzyme of the aerobic denitrification process, intracellular nitrate reductase (Nap), from functioning properly [13]. The gel beads can protect the activity of aerobic denitrifying bacteria and reduce the impact of unfavorable external conditions, hence the smaller influence of temperature and pH changes on the NO3−-N removal efficiency.

3.4. Immobilized Aerobic Denitrifying Bacteria Enhanced the SND Capability of the BSIS

The study investigated the effect of immobilized aerobic denitrifying bacteria on the simultaneous nitrification and denitrification (SND) performance of the BSIS using three reactors: R1 (BSIS), R2 (BSIS + free aerobic denitrifying bacteria), and R3 (BSIS + immobilized aerobic denitrifying bacteria). Figure 5 shows the removal performance of organic matter by R1, R2, and R3. The addition of free and immobilized aerobic denitrifying bacteria had a minor impact on the BSIS’s own CODCr. removal capacity, which might be due to the system maintaining an aerobic state during operation, allowing for the thorough degradation of CODCr. The average CODCr. removal rates after stable operation for R1, R2, and R3 were 91.79 ± 0.8%, 91.82 ± 0.8%, and 91.88 ± 0.6%, respectively.
Figure 6a–c shows the removal performance of NH4+-N, NO3−-N, and NO2−-N. The average effluent NH4+-N concentrations for R1, R2, and R3 were 9.45 ± 1.60 mg/L, 8.28 ± 1.29 mg/L, and 7.51 ± 1.15 mg/L, respectively, and the effluent NO2−-N concentrations were 0.83 ± 0.23 mg/L, 0.94 ± 0.28 mg/L, and 0.78 ± 0.30 mg/L, respectively. The addition of aerobic denitrifying bacteria promoted the complete nitrification of NH4+-N [51]. From days 1 to 5, the microorganisms in R1, R2, and R3 were in the adaptation phase, and the nitrification activity was not high. After the microorganisms adapted to the environment, the activity of the related enzymes increased, and NO2−-N was quickly converted into other forms of nitrogen, with no accumulation of NO2−-N [52]. The effluent NO2−-N in R1, R2, and R3 basically stabilized by day 10, with the effluent NO2−-N concentration essentially at 0.01 mg/L, indicating that the nitrification reaction was thorough and there was no accumulation of NO2−-N [53].
The effluent NO3−-N concentrations from R1, R2, and R3 showed significant differences. The effluent NO3−-N concentration in R1 gradually increased, reaching a maximum of 25.49 ± 1.40 mg/L. The analysis suggests that the reason might be that R1 did not add aerobic denitrifying bacteria, resulting in the weak denitrification capacity of the system and causing substantial accumulation of NO3−-N. The NO3−-N accumulation in R2 and R3 was significantly less than that in R1. From days 1 to 6, the effluent NO3−-N concentration in R2 was lower than that in R3. The analysis indicates that this could be due to the mass transfer resistance of the immobilized gel beads, which required some time for the immobilized aerobic denitrifying bacteria to perform their function effectively, whereas the free aerobic denitrifying bacteria could directly participate in the reaction. Starting from day 7, the effluent NO3−-N concentration in R3 was significantly lower than that in R2, and during days 7–30, the average effluent NO3−-N concentration in R3 was 1.84 ± 0.94 mg/L, while in R2, it was 11.25 ± 2.11 mg/L. The analysis suggests that the immobilization material provided a protective effect, allowing the aerobic denitrifying bacteria to grow and reproduce more effectively and enhancing its resilience to external shock loads. The free aerobic denitrifying bacteria were more prone to loss, resulting in a higher effluent NO3−-N concentration during the later stages of operation. Meanwhile, the effluent NO3−-N concentrations for R1, R2, and R3 were 18.88 ± 2.28 mg/L, 9.52 ± 2.00 mg/L, and 2.43 ± 1.06 mg/L, respectively, indicating that the addition of both free and immobilized aerobic denitrifying bacteria strengthened the system’s denitrification capacity to some extent, and the addition of immobilized aerobic denitrifying bacteria could further enhance the aerobic denitrification capacity of the BSIS itself, which resulted in enhanced SND performance.
Figure 6d shows the variation in the influent and effluent total nitrogen (TN) concentrations and removal rates in each reactor, and there were significant differences in the TN removal efficiency among the reactors. During the initial stage of operation (days 1–10), R2 had the highest TN removal efficiency, which was superior to R3. The analysis suggests that this could be due to the fact that the aerobic denitrifying bacteria added into R2 were in a free state, whereas the immobilized material had a high mass transfer resistance, and the immobilized aerobic denitrifying bacteria needed some time to fully exert their denitrification effect, resulting in R2 having a better TN removal efficiency than R3 at the beginning of the reaction. When the operation stabilized, the average TN removal rates for R1, R2, and R3 were 50.49 ± 6.0%, 70.31 ± 4.0%, and 92.86 ± 2.0%, respectively, and the average TN removal rate of R3 was 22.55 ± 2.0% higher than that of R2, indicating that the immobilization method effectively retained the abundance of the aerobic denitrifying bacteria, preventing them from being lost as the operation time increased. As the operation time grew, the TN removal rate of R3 steadily increased and remained above 95.10 ± 1.0%, demonstrating that the immobilized aerobic denitrifying bacteria could effectively enhance the SND capability of the BSIS.
The experimental results indicate that R1, R2, and R3 all have relatively high nitrogen removal efficiency, which also confirms that the BSIS itself possesses a good simultaneous nitrification and denitrification (SND) capability. The addition of immobilized aerobic denitrifying bacteria further enhanced the SND performance of the BSIS.

4. Conclusions

In this study, three strains of aerobic denitrifying bacteria with good nitrification and denitrification performance were isolated from a BSIS. The isolated aerobic denitrifying bacteria were immobilized in PVA–SA gel beads, using microbial immobilization technology. When the concentrations were PVA 10 g/100 mL, SA 4 g/100 mL, CaCl2 2 g/100 mL, and the amount of bacteria encapsulated was 2 g/100 mL, the immobilized aerobic denitrifying bacteria gel beads could achieve a NO3−-N removal rate as high as 91.73%. The optimal process conditions for nitrogen removal were a shaker speed = 109.24 rpm, a temperature = 23.6 °C, and s pH = 7.9. The experimental results for R1, R2, and R3 showed that the immobilized aerobic denitrifying bacteria could effectively promote the SND performance of the BSIS. During the stable operation period, the average effluent NO3−-N concentration in R3 was 1.53 ± 0.83 mg/L, which was much lower than that of R1 and R2, indicating that the immobilization technology effectively retained the abundance of the aerobic denitrifying bacterial community, allowing it to better exert its aerobic denitrification effect and effectively enhancing the SND capability of the BSIS. Immobilized aerobic denitrifying bacteria technology has broad prospects for large-scale application. The preparation of PVA–SA gel beads is simple, low cost, and has good stability. They can be used to effectively avoid biological loss, reduce operating costs, and they have strong adaptability. Such technology conforms to national energy conservation and emission reduction policies and is an effective means to enhance the denitrification efficiency of the BSIS.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17091308/s1, Table S1: Composition of BTB solid medium. Table S2: Water quality testing methods. Table S3: Preliminary experiment on optimization of the ratio of immobilized materials.

Author Contributions

Conceptualization, J.L. (Jie Li) and Y.W.; methodology, J.L. (Jing Li) and H.M.; software, J.L. (Jing Li) and H.M.; validation, J.L. (Jing Li), H.X. and W.Z.; formal analysis, J.L. (Jing Li) and H.M.; investigation, J.L. (Jing Li); resources, J.L. (Jing Li); data curation, J.L. (Jing Li) and H.M.; writing—original draft preparation, J.L. (Jing Li); writing—review and editing, J.L. (Jing Li); visualization, J.L. (Jing Li) and H.M.; supervision, J.L. (Jie Li) and Y.W.; project administration, J.L. (Jie Li) and J.L. (Jing Li); funding acquisition, J.L. (Jie Li) and J.L. (Jing Li). All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 51768032). The work was also financially supported by the Gansu Provincial Department of Science and Technology Natural Science Foundation (No. 24JRRA234).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

References

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Water 17 01308 g001
Figure 1. Comparison of denitrification performance among aerobic denitrifying bacteria.
Figure 1. Comparison of denitrification performance among aerobic denitrifying bacteria.
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Figure 2. Analysis of the significance of the pH, rotation speed, and temperature on the nitrate removal efficiency. ** Indicates p-value < 0.001.
Figure 2. Analysis of the significance of the pH, rotation speed, and temperature on the nitrate removal efficiency. ** Indicates p-value < 0.001.
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Figure 3. Residual distribution of the fitting model.
Figure 3. Residual distribution of the fitting model.
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Figure 4. Response surface and corresponding contours of NO3−-N removal efficiency of immobilized gel spheres: (a) shaking speed and temperature; (b) shaking speed and pH; and (c) temperature and pH.
Figure 4. Response surface and corresponding contours of NO3−-N removal efficiency of immobilized gel spheres: (a) shaking speed and temperature; (b) shaking speed and pH; and (c) temperature and pH.
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Figure 5. The concentration and removal rate of CODcr in the inlet and effluent during the operation of each reactor.
Figure 5. The concentration and removal rate of CODcr in the inlet and effluent during the operation of each reactor.
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Figure 6. Nitrogen removal for each reactor: (a) NH4+-N; (b) NO2−-N; (c) NO3−-N; and (d) TN. (R1: BSIS; R2: BSIS + free aerobic denitrifying bacteria; R3: BSIS + immobilized aerobic denitrifying bacteria).
Figure 6. Nitrogen removal for each reactor: (a) NH4+-N; (b) NO2−-N; (c) NO3−-N; and (d) TN. (R1: BSIS; R2: BSIS + free aerobic denitrifying bacteria; R3: BSIS + immobilized aerobic denitrifying bacteria).
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Table 1. The results of the preliminary experiment on the optimization of the ratio of immobilized materials.
Table 1. The results of the preliminary experiment on the optimization of the ratio of immobilized materials.
Test NumberSA Dosage (g/100 mL)PVA Dosage (g/100 mL)CaCl2 Dosage (g/100 mL)Bacterial Community Embedding Amount (g/100 mL)NO3−-N Removal Rate (%)
13(1)9(1)2(1)1(1)82.43
23(1)10(2)4(3)3(3)90.15
33(1)11(3)3(2)2(2)84.83
44(2)9(1)3(2)3(3)87.35
54(2)10(2)2(1)2(2)91.73
64(2)11(3)4(3)1(1)82.72
75(3)9(1)4(3)2(2)87.54
85(3)10(2)3(2)1(1)84.33
95(3)11(3)2(1)3(3)82.58
K1257.41257.32256.74249.48
K2261.8266.21256.51264.1
K3254.45250.13260.41260.08
k185.8085.7785.5883.16
k287.2788.7485.5088.03
k384.8283.3886.8086.69
R2.455.361.304.87
Table 2. Response surface experiment results.
Table 2. Response surface experiment results.
Test NumberShaking Speed (rpm)Temperature (°C)pHNO3−-N Removal Rate (%)
150(−1)10(−1)7(0)59.46
2150(1)10(−1)7(0)74.51
350(−1)30(1)7(0)62.44
4150(1)30(1)7(0)78.15
550(−1)20(0)5(−1)63.84
6150(1)20(0)5(−1)80.34
750(−1)20(0)9(1)66.26
8150(1)20(0)9(1)80.45
9100(0)10(−1)5(−1)73.12
10100(0)30(1)5(−1)79.65
11100(0)10(−1)9(1)77.16
12100(0)30(1)9(1)82.54
13100(0)20(0)7(0)88.62
14100(0)20(0)7(0)89.15
15100(0)20(0)7(0)88.43
16100(0)20(0)7(0)88.34
Table 3. The results of the variance analysis of the fitting model.
Table 3. The results of the variance analysis of the fitting model.
Variance SourceSum of SquaresDegrees of FreedomMean SquareF-Valuep-ValueSignificance
Model1555.759172.86191.49<0.0001**
X1–Shaking Speed472.011472.01522.89<0.0001**
X2–Temperature42.92142.9247.550.0002**
X3–pH11.19111.1912.390.0097
X1X20.108910.10890.12060.7386
X1X31.3311.331.480.2635
X2X30.330610.33060.36630.5641
X12679.491679.49752.73<0.0001**
X22224.901224.90249.14<0.0001**
X3243.82143.8248.540.0002**
Residual6.3270.9027
Lack of Fit5.9231.9719.710.0074*
Pure Error0.400340.1001
Total Deviation1562.0716
R2 = 0.9960Signal-to-Noise Ratio = 40.9559
Note: * Indicates 0.001 < p-value < 0.05; ** Indicates p-value < 0.001.
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Li, J.; Li, J.; Wang, Y.; Mu, H.; Xie, H.; Zhao, W. Optimizing the Nitrogen Removal Efficiency of an Intermittent Biological Sponge Iron Reactor by Immobilizing Aerobic Denitrifying Bacteria in the Biological Sponge Iron System. Water 2025, 17, 1308. https://doi.org/10.3390/w17091308

AMA Style

Li J, Li J, Wang Y, Mu H, Xie H, Zhao W. Optimizing the Nitrogen Removal Efficiency of an Intermittent Biological Sponge Iron Reactor by Immobilizing Aerobic Denitrifying Bacteria in the Biological Sponge Iron System. Water. 2025; 17(9):1308. https://doi.org/10.3390/w17091308

Chicago/Turabian Style

Li, Jing, Jie Li, Yae Wang, Hao Mu, Huina Xie, and Wei Zhao. 2025. "Optimizing the Nitrogen Removal Efficiency of an Intermittent Biological Sponge Iron Reactor by Immobilizing Aerobic Denitrifying Bacteria in the Biological Sponge Iron System" Water 17, no. 9: 1308. https://doi.org/10.3390/w17091308

APA Style

Li, J., Li, J., Wang, Y., Mu, H., Xie, H., & Zhao, W. (2025). Optimizing the Nitrogen Removal Efficiency of an Intermittent Biological Sponge Iron Reactor by Immobilizing Aerobic Denitrifying Bacteria in the Biological Sponge Iron System. Water, 17(9), 1308. https://doi.org/10.3390/w17091308

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