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Article

Evolution of Microbial Community Structure and Denitrifying Functional Microorganisms in the Biological Sponge Iron System

by
Jing Li
,
Huina Xie
,
Wei Zhao
* and
Jie Li
*
College of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2025, 15(13), 7244; https://doi.org/10.3390/app15137244 (registering DOI)
Submission received: 23 May 2025 / Revised: 24 June 2025 / Accepted: 25 June 2025 / Published: 27 June 2025
(This article belongs to the Section Ecology Science and Engineering)
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Abstract

With the growing problem of global water pollution, nitrogen pollution has become a key factor affecting aquatic ecosystems and human health. The biological sponge iron system (BSIS) has gained attention as a research hotspot due to its efficient denitrification capability. This study focused on the changes in microbial community structure and the relative abundance and interrelationships of nitrogen cycle-related functional bacteria at different operational stages of the BSIS with a sponge iron (SFe) dosage of 90 g/L. The results showed that as the operation time of the reactor extended, the relative abundance of denitrifying genera such as Saccharimonadales, Arenimonas, and Acinetobacter significantly increased, while the relative abundance of Proteobacteria showed a trend of initial increase followed by a decrease. The relative abundance of nitrifying bacteria exhibited a more complex variation, whereas the abundance of denitrifying bacteria showed a continuous upward trend. In addition, there were complex interrelationships among different denitrifying bacteria, such as a positive correlation between Saccharimonadales and Acetobacteraceae, and a negative correlation between Rhodothermus and Pseudoxanthomonas. This study not only revealed the changes in the relative abundance and interrelationships of microbial communities and nitrogen cycle-related functional bacteria over time with an SFe dosage of 90 g/L, but also provided a new perspective for understanding the intrinsic mechanism of enhanced biological denitrification by sponge iron. These findings are of great significance for optimizing the operating parameters of the BSIS, improving denitrification efficiency, and promoting the practical application of this technology in the field of environmental engineering.

1. Introduction

The surge in demand for food, water, and energy, driven by the expansion of the global economy and a growing population [1], has led to an increase in nitrogen pollution in water bodies [2,3]. Ammonium nitrogen (NH4+-N) [4] and nitrate nitrogen (NO3−-N) pollutants contribute to eutrophication and pose significant risks to human health [5]. While both chemical and biological methods are used for wastewater denitrification, biological methods are generally favored due to their efficiency and cost-effectiveness [6]. Traditional biological denitrification technologies, including the anaerobic–anoxic–oxic (A2/O) process [7], sequencing batch reactor (SBR) systems [8], and the oxidation ditch [9], are challenged by issues such as inconsistent performance and high energy consumption [10].
Iron is an essential trace element for microbial growth, playing a crucial role in promoting microbial proliferation [11]. It is also a component of key enzymes [12], further stimulating microbial activity during enzyme synthesis [13]. Additionally, iron is a vital part of electron transfer cytochromes [14], which are indispensable carriers for intracellular oxidative electron transfer in microbial cells [15]. Iron is increasingly used in wastewater treatment, with zero-valent iron being the most prominent form. The combination of zero-valent iron and microorganisms not only meets the energy needs of the microorganisms but also enhances their environmental adaptability and treatment efficiency [16]. Deng [17] found in their study on the characteristics and mechanism of the influence of organic carbon on Fe(0)-AD that the addition of zero-valent iron promoted the enrichment of iron-oxidizing bacteria (such as Sphaerotilus and Ferriphaselus) and autotrophic denitrifying bacteria (such as Hydrogenophaga and Hyphomicrobium), thereby significantly enhancing the denitrification effect. Zhang [18] found in their study on the influence of zero-valent iron on microbial nitrate removal that under the coexistence of zero-valent iron and microorganisms, nitrate could be almost completely removed, and the addition of zero-valent iron promoted the enrichment of denitrifying functional bacteria such as Methylotenera spp. and Pseudomonas spp. Li [19] used iron–polyurethane foam composite carriers as fillers to investigate the microbial community structure in a sequencing batch activated sludge reactor and found that the functional microorganisms had a relatively high abundance of aerobic heterotrophic denitrifying bacteria, Rhodobacter, and anoxic heterotrophic denitrifying bacteria, Haliangium. Chen [20] added iron chips to a sequencing batch activated sludge reactor on simultaneous nitrification and denitrification (SND), and results showed that iron chips significantly improved nitrogen removal and increased the relative abundance of aerobic denitrifying bacteria, such as Thauera, Thermomonas, Rhodobacter, and Hyphomicrobium, within the SND system.
Sponge iron (SFe) is a special type of zero-valent iron (ZVI) with a loose and porous structure, mainly composed of ZVI (accounting for more than 90%) and containing a small amount of carbon and other impurities [21]. Due to its large specific surface area and high chemical activity, sponge iron is widely used in the field of water treatment [22]. The biological sponge iron system (BSIS) is a composite system formed by introducing SFe into activated sludge in a specific manner [23], which organically combines iron–carbon micro-electrolysis (IC-ME) and the activated sludge process [24]. During the corrosion process of sponge iron, Fe2+ is released. Numerous studies have shown that (1) Fe2+ can promote the synthesis of microbial denitrification-related enzymes and further activate the activity of microorganisms themselves [11], and (2) as the active center of NADH dehydrogenase, ubiquinone, cytochrome c, and cytochrome bc1, the interconversion between Fe2+ and Fe3+ plays a key role in the cellular electron transport chain [25]. In previous studies, sponge iron and polyurethane foam composite materials were co-packed into an aerobic sequencing batch reactor to construct a BSIS. The results showed that this system could achieve simultaneous nitrification and denitrification. High abundances of organic matter-degrading bacteria, aerobic denitrifying bacteria, anoxic denitrifying bacteria, and iron-related autotrophic denitrifying bacteria were detected in the mixed-liquor sludge samples, which significantly enhanced the treatment efficiency of the reactor. In addition, the presence of anoxic and anaerobic zones within the reactor was detected using a dissolved oxygen probe [19].
Previous studies have also demonstrated that varying dosages of sponge iron exert different influences on the microbial community structure within intermittent reactors. Zhu [26] investigated the impact of adding different dosages (0, 45, 90, and 135 g/L) of sponge iron on the microbial community in an intermittent reactor. The results showed that the addition of sponge iron significantly promoted the growth of denitrifying bacteria and iron bacteria, with the 90 g/L dosage having the most pronounced effect on the promotion of denitrifying bacteria. Nevertheless, the dynamic changes in the microbial community and the fluctuations in the relative abundance of functional microorganisms throughout the operational phases of the BSIS system remain to be fully elucidated.
Therefore, this study used 90 g/L of sponge iron (SFe) as a benchmark to conduct an in-depth analysis of the dynamic changes in the microbial community structure within the BSIS across different operational cycles using high-throughput sequencing technology. Furthermore, we explored the potential impact of various operational phases on denitrifying functional microorganisms. These analytical results elucidate the denitrification mechanism of the BSIS. This research provides a robust theoretical framework for the application of sponge iron in biological denitrification processes and lays a solid foundation for the practical implementation of this technology in the field of environmental engineering.

2. Materials and Methods

2.1. Experimental Materials

SFe: The sponge iron used in this experiment was purchased from a company in Beijing. Before use, it was first cleaned with a 10% NaOH solution for 10 min to remove surface grease, followed by cleaning with a 2% sulfuric acid solution to remove surface oxides. Finally, it was repeatedly rinsed with deionized water until the pH of the supernatant was neutral.
Inoculated sludge: The inoculated sludge used in the experiment was taken from the Qilihe Wastewater Treatment Plant in Lanzhou City and used after acclimation treatment with campus domestic wastewater. Before use, the sludge was centrifuged for 5 min at 4000 rpm and weighed. Table S1 shows the sludge volume index (SV), mixed-liquor suspended solids (MLSS), and mixed-liquor volatile suspended solids (MLVSS) of the activated sludge used in the experiment.

2.2. 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, specifically adding 3 mL of milk and 3 mL of inorganic salts to each liter of tap water. The specific formula for the inorganic salts is as follows: (NH4)2SO4: 105 g/L, K2CO3: 64 g/L, NaHCO3: 32 g/L, K2HPO4: 9.728 g/L, and KH2PO4: 3.84 g/L.

2.3. Experimental Setup

R1 (conventional activated sludge system) and R2 (BSIS system) were initiated in two plexiglass reactors, each with a total volume of 5 L and an effective volume of 4 L. The inoculated sludge concentration was 2.5 g/L, with aeration oxygen supplied from the bottom. R2 filled 360 g of SFe into 12 polyurethane foam balls, each with a diameter of 4 cm (SFe content is 90 g/L) [21,26], as shown in Figure 1.

2.4. Test Method

R1 and R2 were started in parallel, and the trends of CODCr, NH4+-N, NO2−-N, NO3−-N, and TN concentrations in the influent and effluent during the operation of the two systems were compared. Each reactor was continuously operated in cycles according to the sequence of influent (15 min), aeration (11 h), sedimentation (30 min), and effluent (15 min), with a water exchange ratio of 1/2 and an accumulated operation of 30 days, for a total of 60 operating cycles. CODCr, NH4+-N, NO3−-N, NO2−-N, TN were measured according to the specific standard methods [27] (Table S2). Data were plotted using Origin 2025. All the experiments were repeated three times, and the data are expressed as the mean ± standard deviation.

2.5. Analysis of Microbial Community Composition

Using the conventional activated sludge system (R1, L1) as a control, activated sludge suspension samples (10 mL) were collected from the system with a sponge iron dosage of 90 g/L (R2) at the initial stage of operation (L2), the stable operation period (L3), and the end of the operation (L4). To reduce the variability of individual samples and improve representativeness, three independent samples were collected at each time point and then mixed into a composite sample for subsequent microbial community analysis. These samples were then mixed and centrifuged, and the supernatant was removed. Before DNA extraction, these four sludge samples were stored in a freezer (−80 °C). DNA sequencing was performed using the Illumina MiSeq sequencing platform from Shanghai Personal Biotechnology Co., Ltd., (Shanghai, China) [19]. Based on the distribution of unrarefied ASV/OTU in different samples, the alpha diversity of each sample was assessed; using the rarefied ASV/OTU table, the beta diversity of each sample was evaluated, and we conducted principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) using R (4.4.1.).

3. Results and Analysis

3.1. Study on the CODCr Removal Performance of a Biological Sponge Iron Reactor

The trends of influent and effluent CODCr concentrations and their removal rates during the operation of R1 and R2 are shown in Figure 2. When comparing the CODCr removal efficiency, it was found that at an average influent CODCr concentration of 301.45 mg/L, the average effluent CODCr concentrations of R1 and R2 were 119.03 ± 3.81 mg/L and 59.93 ± 2.62 mg/L, respectively, with average CODCr removal rates of 60.49 ± 0.23% and 79.94 ± 0.48%, respectively. The average CODCr removal rate of R2 was substantially higher than that of R1, which can be attributed to the addition of SFe that enhanced the catalytic activity of dehydrogenase in the system, thereby accelerating the dehydrogenation reaction of organic matter and promoting the rate of biochemical decomposition of organic matter, resulting in improved CODCr removal efficiency.

3.2. Study on the Nitrogen Removal Performance of a Biological Sponge Iron Reactor

The trends of various nitrogen species during the operation of R1 and R2 are shown in Figure 3. The average influent NH4+-N concentration for R1 and R2 was 49.44 mg/L, with average effluent NH4+-N concentrations of 12.39 ± 1.20 mg/L and 9.45 ± 1.60 mg/L, respectively, corresponding to average NH4+-N removal rates of 74.97 ±0.02% and 80.93 ± 0.03%. This indicates that under long-term aerobic conditions, SFe addition enhanced autotrophic nitrifier activity, facilitating the conversion of NH4+-N to NO3−-N or other nitrogen oxides. Initially, R2 showed an accumulation of NO2−-N, which can be attributed to NO2−-N being an intermediate product of nitrification that is easily degraded but requires a longer reaction time. In the early stages of R2 operation, the microorganisms were still adapting to the environment, and nitrification was not yet fully activated. As the microorganisms gradually adapted to the environment and enzyme activities increased, NO2−-N was rapidly converted to other forms of nitrogen, and NO2−-N no longer accumulated [28]. R1, having already adapted to the system environment during previous long-term cultivation, exhibited less fluctuation in effluent NO2−-N concentration. Ultimately, the effluent NO2−-N concentrations in both R1 and R2 stabilized, with effluent NO2−-N concentrations basically at 0.01 mg/L, indicating that nitrification was thorough and NO2−-N did not accumulate substantially. The effluent NO3−-N concentration in R2 was substantially lower than that in R1. The reason for this could be that iron, as an essential trace element for microbial growth and metabolism, substantially promotes the physiological activities of nitrifying and denitrifying bacteria, thereby effectively mitigating the accumulation of NO3−-N during simultaneous nitrification and denitrification. The average effluent TN concentrations for R1 and R2 were 34.39 ± 1.76 mg/L and 29.16 ± 3.03 mg/L, respectively, with average TN removal rates of 30.45 ± 0.03% and 41.05 ± 0.06%. The TN removal efficiency of R2 was substantially better than that of R1. This superior denitrification performance can be attributed to the important role of iron in microbial metabolism. The incorporation of SFe provided the system with abundant zero-valent iron (Fe0), Fe2+, and Fe3+, which greatly promoted the development and proliferation of the microbial community, enhancing the biodiversity and metabolic functions of the system. The oxygen-consuming corrosion of SFe created a gradient environment ranging from microoxic to anaerobic on its surface and in adjacent areas [29], providing favorable ecological niches for the coexistence of aerobic and anaerobic microorganisms. Additionally, Fe0 can undergo electrochemical, hydrolysis, redox, and Fenton reactions, further enhancing the denitrification performance of R2.
Overall, R2 exhibited significantly better nitrogen removal capacity than R1, indicating that the addition of SFe enhanced the denitrification efficiency of activated sludge. The reasons for this improvement are likely due to SFe being a porous material that provides microenvironments for aerobic, facultative, and anaerobic microorganisms, which are conducive to microbial growth and metabolism. Additionally, iron, as an essential element for cell growth, not only promotes the acceleration of electron transfer rates but also enhances the activity of dehydrogenase. The combined effects of these factors substantially improve the denitrification efficiency.

3.3. The Impact of Sponge Iron on the Microbial Community Structure

3.3.1. Analysis of Microbial Community Richness and Diversity

The Simpson [30] index and Shannon [31] index are important indicators for measuring the diversity of microbial species. An increase in these indices means that the types of microorganisms in the sample are more diverse. The Chao1 index [32] and ACE index [33], on the other hand, are used to reflect the species richness of the microbial community. An increase in their values indicates an improvement in the richness of the microorganisms. Generally, the Chao1 index is significantly affected by the number of low-abundance species, while the ACE index reduces this bias by setting the abundance threshold for rare species to 10. The Alpha diversity indices of mixed-liquor sludge samples from each reactor are shown in Table 1.
From Table 1, it can be seen that the Shannon indices for each sludge sample are 8.46 (L1), 7.64 (L2), 8.25 (L3), and 7.32 (L4); and the Simpson indices are 0.99 (L1), 0.97 (L2), 0.98 (L3), and 0.96 (L4), respectively. It is found that both the Shannon and Simpson indices in the L2–3 samples are lower than those in the L1 sample, indicating that the addition of 90 g/L SFe reduces the microbial diversity in the reactor, and microorganisms sensitive to iron ion concentration are eliminated. The L3 sample is higher than the L2 and L4 samples, indicating that after a period of operation, iron bacteria and denitrifying bacteria proliferate in the reactor, increasing microbial diversity. However, by the end of the operation, the excessively high accumulated iron ion concentration in the reactor inhibits the growth and reproduction of microorganisms. Compared with L1, the Chao1 and ACE indices of L2, L3, and L4 increased significantly, indicating that the addition of SFe can effectively promote the richness of microorganisms in the reactor.
As shown in Figure 4, the microbial diversity analysis of the four mixed-sludge samples identified a total of 13,327 operational taxonomic units (OTUs). Specifically, the number of unique OTUs in the samples from the conventional activated sludge system (L1) and the bio-iron sponge system (BSIS) during the start-up phase (L2), stable-operation phase (L3), and end-of-operation phase (L4) were 2020, 2936, 4646, and 3725, respectively. Notably, the number of OTUs shared among these four systems was only 210. The phenomenon where the number of unique OTUs was significantly higher than that of shared OTUs revealed significant differences in the microbial community structure among the systems. This result clearly indicates that, although there is limited overlap in microbial composition among different samples, the introduction of iron sponge (SFe) significantly promoted the enrichment and succession of new microbial groups, thereby enhancing the diversity level of the entire microbial community at the OTU level.
The analysis results indicate that PCoA can explain 75.9% of the variance in the microbial community. By examining Figure 5a, it can be seen that L3 and L4 are closely adjacent on the coordinate axis, while L1 and L2 are more dispersed relative to L4. This result suggests that in the BSIS reactor, the microbial community structure in the mixed-liquor sludge during the middle (L3) and late (L4) stages of operation exhibits a high degree of similarity in the dimensions examined. In contrast, the microbial community composition of the conventional activated sludge system (L1) shows lower similarity to that of the BSIS system. From Figure 5b, it can be observed that, compared to the proximity of sludge samples (L2 to L4) from the BSIS system with added SFe, the control-group sludge sample without SFe (L1) appears relatively isolated. This phenomenon further reveals significant differences in the microbial communities between the two systems. Additionally, the distance between L3 and L4 is shorter than the distances between L1 and L3 and L1 and L4, indicating significant differences in the microbial community structure between the initial-operation phase and both the stable-operation phase and the final-operation phase. A possible explanation for this difference is that in the reactor with 90 g/L SFe added, microorganisms are gradually adapting to environmental changes in the initial-operation phase, and iron-reducing bacteria (IRB) and iron-oxidizing bacteria (IOB) begin to proliferate. In the stable-operation phase, these iron bacteria have formed a stable biofilm structure and occupy the dominant ecological niche in the system.

3.3.2. Analysis of the Microbial Community Structure

The analysis of the microbial structure and hierarchical clustering of the top 10% relative abundance at the phylum level in the conventional activated sludge system and the BSIS system with an SFe dosage of 90 g/L (initial operation L2, stable operation L3, and final operation L4) is shown in Figure 6. The results indicate that there are differences in the microbial community composition between the conventional activated sludge system and the BSIS system, suggesting that the addition of SFe can alter the microbial community structure at the phylum level. The microbial community composition of the BSIS system during the stable-operation phase (L3) and the final-operation phase (L4) is highly similar, which is consistent with the conclusions of the beta diversity analysis. In L1–L4, the top ten phyla by relative abundance include Proteobacteria, Patescibacteria, Bacteroidetes, Chloroflexi, Acidobacteria, Actinobacteria, Nitrospirae, and some other phyla with relatively low abundance.
Proteobacteria has the highest relative abundance in all samples, accounting for 44.35% in the stable conventional activated sludge system (L1), and 57.35%, 47.52%, and 43.50% in the BSIS system sludge during the initial-operation phase (L2), stable-operation phase (L3), and final-operation phase (L4), respectively. L2 and L3 increased by 13.00% and 3.17% compared to L1, while L4 was slightly lower by 0.85% compared to L1. Proteobacteria is an important genus in the process of denitrification and nitrite reduction [34], including various nitrogen-fixing, nitrifying, and denitrifying bacteria [35], which can participate in carbon and nitrogen cycles and decompose organic macromolecules such as carbohydrates and proteins [36]. The lipopolysaccharides on the surface of Proteobacteria are beneficial for microbial attachment and growth and drive activated sludge flocculation [37], playing an important role in biological nitrogen and phosphorus removal and pollutant degradation [38]. In the initial-operation phase of the BSIS system, the relative abundance of Proteobacteria showed an upward trend, which may be due to the fact that iron ions play a vital role in the life cycle of microorganisms, and low concentrations can promote the growth and reproduction of Proteobacteria. In the stable-operation phase and final-operation phase of the BSIS system, the relative abundance of Proteobacteria began to decline, indicating that with the increasing concentration of iron ions, it is not conducive to the enrichment of Proteobacteria, and there was even an inhibitory trend in the final-operation phase. Patescibacteria is the second dominant bacterium in all samples. This bacterium has a synergistic effect with key genes nifH and amoA in the nitrogen cycle and can participate in the ammonia oxidation process [39] with relative abundances of 15.76%, 11.41%, 21.05%, and 33.29%, respectively. Zhang [39] found that Patescibacteria often coexist with denitrifying bacteria in groundwater. Zhao [40] found that Patescibacteria were detected in partial nitrification–anammox systems participating in the ammonia oxidation process, and are a typical phylum of the carbon cycle, with a synergistic effect with nitrogen cycle genes nifH and amoA [41]. In the stable-operation phase and final-operation phase of the BSIS system, the relative abundance of Patescibacteria increased, indicating that the addition of SFe is beneficial to the growth and reproduction of Patescibacteria. Bacteroidetes is the third dominant bacterium in all samples, with relative abundances of 23.60%, 19.91%, 10.18%, and 7.76%, respectively. Bacteroidetes can participate in protein decomposition and play an important role in organic matter degradation [42]. However, the addition of SFe reduced the relative abundance of Bacteroidetes, indicating that the growth and reproduction process of Bacteroidetes is sensitive to iron ion concentration, and the higher the iron ion concentration, the worse its activity. Chloroflexi is a facultative anaerobic bacterium that can strengthen the biofilm structure [43] and has a strong effect on organic matter degradation [44,45], with relative abundances of 4.73%, 3.11%, 3.64%, and 4.63%, respectively. This indicates that with the operation of the reactor, a stable anaerobic zone is gradually formed in the BSIS system, providing a good living environment for facultative anaerobic bacteria. Acidobacteria has a high relative abundance in the BSIS system, with 2.43%, 4.72%, and 4.52% in the initial-operation phase, stable-operation phase, and final-operation phase of the BSIS sludge, respectively, all higher than the 0.89% in the conventional activated sludge system. Acidobacteria is a typical heterotrophic microorganism that has the ability of nitrification and aerobic denitrification and can carry out these two processes simultaneously [46,47]. The increase in the relative abundance of Acidobacteria in the BSIS system indicates that SFe can promote its growth and reproduction, and the “anaerobic–anoxic–aerobic” microenvironment created by the addition of SFe SPBF is beneficial for the interaction between heterotrophic nitrifying aerobic denitrifying microorganisms and various nitrogenous substances. Actinobacteria have been proven to have a denitrification effect under anaerobic conditions [48], and their relative abundance in the BSIS system (3.04%, 1.72%, and 1.68%) is lower than that in the conventional activated sludge system (4.88%), indicating that the addition of SFe inhibits the growth and reproduction of Actinobacteria. Nitrospirae contains many nitrifying bacteria with good nitrification ability [49] and has the highest relative abundance in the stable-operation phase of the BSIS system, at 2.30%, indicating that SFe promotes the role of Nitrospirae. The aggregation of these phyla in the reactor with an SFe dosage of 90 g/L is beneficial to the transformation of nitrogen and the removal of organic pollutants in the BSIS system, which is also consistent with the results shown in Figure 2 and Figure 3.
At the genus level (Figure 7), a notable shift was observed in the microbial community from L1 to L4. The reddish-hued groups experienced significant fluctuations, and the relative abundance of certain genera decreased substantially. The most abundant dominant species in the four samples is Saccharimonadales. In the stable conventional activated sludge system and the BSIS system sludge, during the initial-operation phase, stable-operation phase, and final-operation phase, they account for 21.68%, 16.24%, 33.63%, and 38.55%, respectively. Saccharimonadales are known to promote the formation of aerobic granular sludge [50]. There is a positive correlation between the denitrification genes within Saccharimonadales and the removal efficiency of NO3−-N [51], making them common denitrifiers [52]. Within optimal iron concentration ranges, Saccharimonadales are capable of synthesizing requisite iron-dependent enzymes, thereby sustaining efficient metabolic processes and achieving maximal growth rates as well as cellular yields. Chi [53] uncovered a significant positive correlation between the relative abundance of Saccharimonadales bacteria in the rhizosphere soil and the available iron content, suggesting that Saccharimonadales may play a role in the iron cycling within the soil and potentially assist plants in acquiring this essential nutrient. The increase in iron ion concentration substantially promotes the growth and reproduction of Saccharimonadales, which aligns with the trends in the physicochemical properties of the samples in this study. Arenimonas is a hydrogenotrophic denitrifying bacterium that can utilize iron as an electron donor, reducing nitrate to nitrite [54]. Arenimonas can perform hydrogenotrophic denitrification under certain conditions [55], with a relative abundance of 3.19% in the sludge of the BSIS system at the final-operation phase, much higher than in the conventional sludge system and the BSIS system at the initial- and stable-operation phases (1.17%, 0.19%, and 1.60%). The enrichment of Arenimonas in the BSIS system may be attributed to the dissolution of iron ions from the corrosion of sponge iron and the production of cathodic H2, which creates a favorable environment for its growth. Simplicispira, an iron-reducing bacterium with denitrification function, can use hydrogen produced by iron corrosion for denitrification of nitrate and nitrite under both aerobic and anaerobic conditions [56]. Their relative abundances in the stable conventional activated sludge system and the BSIS system sludge during the initial-, stable-, and final-operation phases are 0.24%, 0.64%, 0.16%, and 0.15%, respectively. The relative abundance of Simplicispira was 0.4% higher in the initial phase of SFe addition than in the conventional activated sludge system, indicating that trace amounts of iron ions from SFe corrosion increased the activity of Simplicispira. Nitrospira plays an important role in the nitrification process, with relative abundances of 1.10%, 0.89%, 2.37%, and 1.15%. Acidovorax is a typical nitrate-dependent divalent iron-oxidizing bacterium, and the addition of SFe promotes its growth and reproduction to some extent. The concentration of iron ions has an inhibitory effect on the relative abundances of aerobic denitrifiers such as Rhodobacter, Thauera, and Cupriavidus, while promoting the relative abundances of Leptothrix, Thermomonas, and Defluviimonas. Additionally, the relative abundances of denitrifying bacteria with different functions, such as Haliangium, Runella, OLB12, and CL500-29_marine_group, have all been enhanced to some extent, making the denitrification performance of the BSIS system superior to that of the conventional activated sludge system.

3.3.3. Analysis of Key Functional Bacteria and Their Correlations

To further explore the impact of SFe addition on the dynamics of the microbial community, a detailed analysis of nitrogen cycle-related functional bacteria in each sample was conducted, and the interrelationships among these functional bacteria were investigated.
Figure 8a illustrates the changes in the relative abundances of nitrifying and denitrifying bacteria across L1–L4, along with a correlation analysis. The relative abundance of denitrifying bacteria is significantly higher than that of nitrifying bacteria in all stages. Notably, during the stable- and final-operation phases of the BSIS system, the relative abundance of denitrifying bacteria increased markedly. This enhancement enables a more efficient reduction of nitrates to nitrogen gas, thereby boosting denitrification efficiency. These findings further confirm that the nitrogen removal performance of the reactor with added SFe continues to improve over time. Xiang [57] evaluated the effect and mechanism of sponge iron as an inorganic electron donor combined with the WLASI system on improving water quality in low C/N water source reservoirs. The study found that this coupled system significantly enhanced the diversity and abundance of aerobic denitrifying microorganisms in the water body, with the relative abundance of key genera such as Mycobacterium and hgcI_clade increasing notably. Du [3] constructed three biological filters with different fillers—ceramsite (R-C), an iron–carbon mixture (R-Fe-C), and pure iron–carbon (R-Fe)—and found that the addition of iron–carbon material significantly promoted the enrichment of heterotrophic denitrifying bacteria (DNB) and aerobic denitrifying bacteria (HNADB), particularly increasing the relative abundance of HNADB in the R-Fe reactor by 7.3 times, indicating that iron has a significant promoting effect on the growth of HNAD bacteria.
The changes in the relative abundance of nitrifying bacteria in L1–L4 are shown in Figure 8b. Across the stages L1–L4, the total relative abundance of nitrifying bacteria was 1.06%, 0.92%, 2.496%, and 1.13%, respectively. In the BSIS system with 90 g/L SFe addition, it exhibited a trend of initial decrease, followed by an increase, and then a subsequent decrease. However, at the end of the operation, the overall relative abundance remained higher than that in the conventional activated sludge system. Nitrospira, Nakamurella, and SM1A02 are the dominant nitrifying genera. Notably, Nitrospira exhibits the highest relative abundance in all samples, with values of 0.89%, 0.79%, 2.09%, and 1.04%, respectively. Nitrospira is commonly found in various natural environments [58], especially in agricultural soils, lakes, rivers, and underwater sediments, and possesses complete ammonia oxidation capability [45]. Nakamurella is a newly classified taxon under the phylum Actinobacteria, with short, rod-shaped cells and non-pathogenic properties. This genus plays a crucial role in achieving stable denitrification and promoting nitrite oxidation reactions [59]. The relative abundance of nitrifying bacteria in sample L3 is significantly higher than in the other three samples, indicating that even when the abundance of nitrifying bacteria is lower than that of denitrifying bacteria, the addition of SFe can still effectively promote the growth of nitrifying bacteria and accelerate nitrification reactions. However, as the operation process nears its end, the abundance of nitrifying bacteria decreases to the initial level, which may be due to the bio-toxicity of the accumulated iron ions in the reactor, thereby inhibiting their growth. Additionally, according to the analysis in Figure 9a, there is a positive correlation between Nitrospira and SM1A02. In contrast, Nakamurella shows a significant negative correlation with both Nitrospira and SM1A02, which may be attributed to the inhibitory effect of SFe.
The changes in the relative abundance of denitrifying bacteria in L1–L4 are shown in Figure 8c. In L1–L4, the overall relative abundances of denitrifying bacteria are 32.84%, 24.42%, 39.47%, and 43.90%, respectively. As the reactor continues to operate, the abundance of denitrifying bacteria within the BSIS system shows an increasing trend, indicating that SFe has a significant promoting effect on the aggregation of denitrifying bacteria. The increase in iron ion concentration is positively correlated with the enrichment degree of denitrifying bacteria. During this process, the relative abundances of Saccharimonadales, Arenimonas, and Acinetobacter continue to rise. In particular, Saccharimonadales, a typical denitrifying bacterium within the Patescibacteria phylum, shows an abundance proportional to that of denitrification-related genes [50], reaching a relative abundance of 35.07% in the BSIS system at the end of the operation. Arenimonas, an autotrophic denitrifying bacterium capable of utilizing iron and reducing nitrates, has a relative abundance of 2.90% in the BSIS system at the end of the operation. Acinetobacter, an autotrophic denitrifying bacterium that plays a key role in degrading complex organic matter and removing CODCr, reaches a relative abundance of 0.41% in the BSIS system at the end of the operation. The enrichment of these denitrifying bacteria contributes to the removal of nitrogen in the system. In contrast, Ferruginibacter, Rhodoferax, Rhodobacter, Defluviimonas, and Thauera have significantly lower relative abundances in the BSIS system compared to the conventional activated sludge system, indicating that these species have poor tolerance to iron ions. Ferruginibacter plays a significant role in denitrification and exhibits strong tolerance to compounds such as Zn(II) [60]. Additionally, Rhodoferax is a typical denitrifying bacterium, widely distributed in both biological nitrogen removal and natural systems, and an increase in its relative abundance facilitates the transformation of NO3−-N [61]. Defluviimonas possesses the capability to reduce nitrate to nitrogen gas under aerobic conditions, thereby promoting the occurrence of simultaneous nitrification and denitrification [62]. Thauera represents a denitrifying bacterium frequently encountered within wastewater treatment facilities [63]. The low abundance of these bacteria in the BSIS system also confirms that the iron ion environment may exert a certain selection pressure or inhibitory effect on these species.
By analyzing Figure 9a,b, complex correlations among different denitrifying bacteria can be observed. For example, there is a strong positive correlation between Saccharimonadales and Acetobacteraceae, indicating that these two genera may be interdependent or co-occur under specific environmental conditions. Similarly, a positive correlation exists between Thermomonas and Flavobacterium, suggesting they may share similar niche strategies or competitive relationships. On the other hand, Rhodothermus and Pseudoxanthomonas show a significant negative correlation, indicating that they may compete for resources in different environments or inhibit each other in the same environment. The negative correlation between Hydrogenophaga and Inhella may reflect their competitive relationship under specific environmental conditions.

4. Conclusions

The results of this study indicate that, as the operation time extended, significant changes occurred in the microbial community structure and the relative abundance of denitrifying functional microorganisms within the BSIS reactor dosed with 90 g/L of sponge iron. These changes strongly confirm that the addition of sponge iron can progressively enhance the system’s denitrification performance as the operational cycles increase. This finding not only reveals the crucial role of microbial succession in the sponge iron-enhanced denitrification process but also highlights its immense potential for improving system efficiency, constituting an important innovation point of this study.
The findings of this research provide significant theoretical guidance for optimizing the operating parameters of sponge iron-enhanced SBR systems and maintaining their long-term stable operation. For example, by real-time monitoring of the dynamic changes in key functional microbial communities, operational strategies can be adjusted in a timely manner to ensure the system consistently maintains high-efficiency denitrification performance. Looking ahead, the microbial response mechanisms revealed in this study offer a new perspective for the field. Future research could further explore the patterns of microbial community succession under different sponge iron dosages and operational conditions, as well as how to maximize the enhancement effect of sponge iron through more precise microbial regulation strategies. This will ultimately promote the wider application of this technology.

5. Future Research Directions

In this study, 90 g/L of sponge iron was used as the experimental condition to thoroughly investigate the evolution patterns of the microbial community structure and the relative abundance of functional microorganisms. Future research should focus on further clarifying the mechanisms by which sponge iron affects microbial electron transfer processes, enzyme activities, and the regulation of gene expression related to denitrification. This will lead to a deeper understanding of the interaction between sponge iron and microorganisms, as well as their combined effect on enhancing denitrification performance. Additionally, future studies should conduct long-term operational experiments to assess the long-term stability and economic feasibility of the BSIS, providing strong data support and theoretical guidance for its widespread application.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app15137244/s1, Table S1: Inoculated Activated Sludge Indicators; Table S2: Water quality testing methods.

Author Contributions

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

Funding

This study was 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 no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
SFesponge iron
BSISbiological sponge iron system
NH4+-Nammonium nitrogen
NO3−-Nnitrate nitrogen
NO2−-Nnitrite nitrogen
TNtotal nitrogen
ZVIzero-valent iron
A2/Oanaerobic–anoxic–oxic
SBRsequencing batch reactor
DOdissolved oxygen
PCoAprincipal coordinates analysis
NMDSnon-metric multidimensional scaling

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Figure 1. Schematic diagram of the experimental setup.
Figure 1. Schematic diagram of the experimental setup.
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Figure 2. The concentration and removal rate of CODcr in influent and effluent during the operation of each reactor.
Figure 2. The concentration and removal rate of CODcr in influent and effluent during the operation of each reactor.
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Figure 3. Effects of aerobic starvation on nitrogen removal in each reactor: (a) NH4+-N, (b) NO2−-N, (c) NO3−-N, and (d) TN.
Figure 3. Effects of aerobic starvation on nitrogen removal in each reactor: (a) NH4+-N, (b) NO2−-N, (c) NO3−-N, and (d) TN.
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Figure 4. Venn diagram of each sample.
Figure 4. Venn diagram of each sample.
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Figure 5. Beta diversity analysis. (a) Principal coordinate analysis (PCoA) and (b) non-metric multidimensional scaling (NMDS) analysis.
Figure 5. Beta diversity analysis. (a) Principal coordinate analysis (PCoA) and (b) non-metric multidimensional scaling (NMDS) analysis.
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Figure 6. Microbial analysis at the phylum level for each sample (top 10%): (a) community structure analysis and (b) hierarchical clustering analysis.
Figure 6. Microbial analysis at the phylum level for each sample (top 10%): (a) community structure analysis and (b) hierarchical clustering analysis.
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Figure 7. Microbial analysis at the genus level for each sample (top 20%): (a) community structure analysis and (b) hierarchical clustering analysis.
Figure 7. Microbial analysis at the genus level for each sample (top 20%): (a) community structure analysis and (b) hierarchical clustering analysis.
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Figure 8. Changes in relative abundance: (a) nitrifying/denitrifying bacteria among samples, (b) nitrifying bacteria, and (c) denitrifying bacteria.
Figure 8. Changes in relative abundance: (a) nitrifying/denitrifying bacteria among samples, (b) nitrifying bacteria, and (c) denitrifying bacteria.
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Figure 9. Functional bacteria correlation analysis (a) functional bacteria and (b) denitrifying bacteria. * p <= 0.05.
Figure 9. Functional bacteria correlation analysis (a) functional bacteria and (b) denitrifying bacteria. * p <= 0.05.
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Table 1. Alpha diversity indices of mixed-liquid sludge samples from different reactors.
Table 1. Alpha diversity indices of mixed-liquid sludge samples from different reactors.
Sample NumberSimpsonChao1ACEShannon
L10.99118411848.46
L20.97128612867.64
L30.98253727428.25
L40.96207522877.32
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MDPI and ACS Style

Li, J.; Xie, H.; Zhao, W.; Li, J. Evolution of Microbial Community Structure and Denitrifying Functional Microorganisms in the Biological Sponge Iron System. Appl. Sci. 2025, 15, 7244. https://doi.org/10.3390/app15137244

AMA Style

Li J, Xie H, Zhao W, Li J. Evolution of Microbial Community Structure and Denitrifying Functional Microorganisms in the Biological Sponge Iron System. Applied Sciences. 2025; 15(13):7244. https://doi.org/10.3390/app15137244

Chicago/Turabian Style

Li, Jing, Huina Xie, Wei Zhao, and Jie Li. 2025. "Evolution of Microbial Community Structure and Denitrifying Functional Microorganisms in the Biological Sponge Iron System" Applied Sciences 15, no. 13: 7244. https://doi.org/10.3390/app15137244

APA Style

Li, J., Xie, H., Zhao, W., & Li, J. (2025). Evolution of Microbial Community Structure and Denitrifying Functional Microorganisms in the Biological Sponge Iron System. Applied Sciences, 15(13), 7244. https://doi.org/10.3390/app15137244

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