The study investigated the denitrification effect of the iron autotrophic denitrification process for removing nitrite under anaerobic conditions, utilizing sponge iron as the electron donor. When the C/N ratio equaled 1, defined as the ratio of chemical oxygen demand to total nitrogen (TN), and the influent nitrite nitrogen (NO2-N) was at 80 mg/L, the average steady-state TN effluent concentration of this system was 41.94 mg/L during the 79-day experiment. The TN value exhibited a significant decrease compared to both the sponge iron system (68.69 mg/L) and the carbon source system (56.50 mg/L). Sponge iron is beneficial for providing an electron donor and ensuring an anaerobic system, fostering an environment that promotes microorganism growth while effectively inhibiting the conversion of nitrite to nitrate. In addition, carbon sources play a vital role in ensuring microorganism growth and reproduction, thereby aiding in TN removal. The optimal parameters based on the effectiveness of TN removal in the iron autotrophic denitrification system were determined to be s-Fe0 dosage of 30 g/L and C/N = 1.5. These results suggest that the iron autotrophic denitrification process, driven by sponge iron, can effectively remove nitrite under anaerobic conditions.

  • The feasibility of reducing nitrite by iron autotrophic denitrification driven by sponge iron was verified.

  • The average total nitrogen effluent concentration of the system during the stable period is 41.94 mg/L.

  • The optimal parameters were determined as s-Fe0 dosage 30 g/L and C/N = 1.5.

  • Both sponge iron and carbon source could effectively promote the removal of nitrite.

  • Carbon sources play a more critical role in the reaction system.

Inorganic nitrogen compounds are prevalent water pollutants (Wells et al. 2018). Nitrite accumulation can occur in anaerobic environments via denitrification, resulting in public health concerns (Lim et al. 2018). Nitrite is considered more toxic than nitrate according to the U.S. Environmental Protection Agency (U.S. EPA), which has established a lower standard for maximum contaminant levels in drinking water for nitrite–nitrogen (1 mg/L) compared to nitrate–nitrogen (10 mg/L) (Fan & Steinberg 1996). Therefore, it is essential to investigate the conversion of nitrite in aquatic environments (Tugaoen et al. 2018).

The biological method represents the primary process for removing nitrogen, characterized by high efficiency and low consumption (Wang et al. 2018). The main process of traditional biological denitrogenation of wastewater is nitrification–denitrification: Under aerobic conditions, the nitrogen present in wastewater is oxidized by aeration and biological nitrification ultimately resulting in NO3-N. Afterward, denitrifying bacteria use organic matter as an electron donor to eliminate nitrate from the water in anaerobic conditions (Zhang et al. 2019). Nitrification processes tend to occur readily, whereas denitrification processes necessitate adequate sources of carbon to fulfill microbial requirements for electron donors and nutrients. Low carbon-to-nitrogen (C/N) ratios are prevalent in nitrate-contaminated wastewaters (Šereš et al. 2019), and the absence of electron donors can restrict nitrate removal by microorganisms (Ji et al. 2015; Wang et al. 2015a). The high concentration of nitrate and nitrite inhibits the positive reaction of nitrification, the nitrification reaction cannot be carried out smoothly, and the degradation capacity of ammonia nitrogen will be reduced. To address these issues, microorganisms require supplementary electron donors. The addition of external carbon sources (e.g., acetate (Akunna et al. 1993) and glucose (Guerra-Santos et al. 1984), etc.) may cause secondary contamination (Jiang et al. 2020) and is unfavorable in terms of cost (Wang et al. 2015b). Therefore, chemical nutrient denitrification has received a lot of attention (Wang et al. 2020a). Iron plays an extremely important role in the Earth's chemical cycles with its abundant reserves (the fourth most abundant element in the Earth's crust) and a wide range of valence changes (−2 to +6) (Johnson et al. 2012; Ilbert & Bonnefoy 2013). Iron can serve as an electron donor for microbial nitrate reduction. It is widely available, cost-effective, and less susceptible to secondary contamination than organics. In addition, the Fe(III) produced by the reaction can be utilized for phosphate removal (Zhang et al. 2015). Therefore, the application of iron-based autotrophic denitrification technology in deep treatment or tailwater intensification has high applicability and important practical significance.

As a new type of denitrification technology, iron-based autotrophic denitrification has the advantages of low cost and no secondary pollution, so it has a broad prospect and research potential (Kiskira et al. 2017). Many studies have been reported on iron-based autotrophic denitrification (Liu et al. 2020; Jokai et al. 2021). However, its application in practice is rarely documented. This is mainly because iron-based autotrophic denitrification to obtain higher denitrification efficiency requires the addition of large amounts of Fe(II), but it is currently not possible to quickly and efficiently discharge the generated Fe(III) mineral precipitation in a timely manner. Fe(II) regeneration by in situ regeneration may be an effective means to solve the aforementioned problems. Sponge iron (noted as s-Fe0) is a porous carbon-containing metallized material with zero-valent iron (ZVI) as the main component, with the advantage of low price and easy transportation and storage. The inherent chemical properties and physical configuration allow for a large number of Fe0/C microelectrolytic corrosion system units. Among them, the anode can continuously introduce effective Fe(II) for the nitrate-dependent ferrous oxiation (NDFO) process (as shown in Equation (1) (Yang et al. 2017; Song et al. 2020)). Song et al. (2020) suggested that activated carbon is catalytic and has a high selectivity in converting nitrate to nitrogen. Second, NDFO is an acid-producing process. The addition of sponge iron can be found through reaction 2 to make the cathode consume hydrogen ions or generate hydroxide ions, which can be an effective means to stabilize the efficiency of NDFO. In addition, the presence of iron sponge can also act as a deaerating agent to ensure an anaerobic environment for biological denitrification. However, numerous studies have found that the zero-valent iron in sponge iron can chemically react with nitrate nitrogen to produce ammonia nitrogen and Fe(II) (Equation (2)). It was also found that the high concentration of Fe(II) facilitated the dissimilatory nitrate reduction to ammonium, but inhibited the denitrification process (Cojean et al. 2020). In summary, sponge iron may be a suitable electron donor material for iron-based autotrophic denitrification:
formula
(1)
formula
(2)

When used alone, sponge iron provides a limited electron donor for denitrification. Studies have shown that only a few Fe autotrophic denitrifying strains are recommended for autotrophic growth using only Fe(II) and nitrate (Li et al. 2014), and most such strains require organic compounds as co-substrates (Chakraborty et al. 2011).

In this study, activated sludge from a long-term laboratory operation was inoculated in a reactor, and sponge iron was injected to remove nitrite from wastewater. The concentrations of ammonia, nitrate, and nitrite nitrogen in the influent and effluent were measured; the removal efficiency was calculated, and chemical oxygen demand (COD), TFe, and Fe(Ⅱ) concentrations and pH were observed. The objectives of this study are (1) to evaluate the feasibility of iron-based autotrophic denitrification combined with heterotrophic denitrification using sponge iron as the iron source to treat nitrite in wastewater in a long-running reactor; (2) to analyze the contribution and effect of sponge iron and carbon source in the reaction system; (2) to analyze the contribution and influence of sponge iron and carbon source in the reaction system; and (3) to investigate the optimal sponge iron dosage and C/N ratio under the reaction system. This work is expected to provide a theoretical basis for the further development and utilization of the process, which is of great significance for the control of nitrogen pollution in water bodies.

Seeding sludge, synthetic wastewater, and s-Fe0

The sludge from a biological sponge iron system (Xie et al. 2021) in long-term stable operation in the laboratory was taken and domesticated for 28 days in an anaerobic reactor with a sponge iron dosing rate of 40 g/L, an influent nitrite concentration of 80 mg/L (as N), and a sodium acetate concentration of 0.1026 g/L (C/N = 1), and its stable average total nitrogen (TN) removal rate reached 85.95%. The sludge was taken as the inoculated sludge for this experiment. The inoculated sludge had SS (suspended solids) and VSS (volatile SS) of 16.45 and 6.40 g/L, respectively, SV (sludge settling velocity) of 37%, Fe(II) concentration of 2,659.65 mg/L, and TFe concentration of 3,334.15 mg/L.

The reagents used in this work were all analytically pure (Sinopharm Chemical Reagent Co., Ltd; purity >99%). Synthetic wastewater was used as feed water for the experimental study of iron-based autotrophic denitrification reactor. The synthetic wastewater consisted of macronutrients and trace elements, where the detailed composition and concentration of macronutrients are listed in Table 1, and the composition and concentration of trace elements are referred to the study by Wang et al. (2020b).

Table 1

Synthetic wastewater components

Components and concentrations of the macronutrient
Chemical substanceConcentrationChemical substanceConcentration
NaNO2 0.39 g/L MgSO4·7H20.025 g/L 
KH2PO4 0.1 g/L CaCl2·2H20.025 g/L 
NaHCO3 1.0 g/L Micronutrient 1 mL/L 
Components and concentrations of the macronutrient
Chemical substanceConcentrationChemical substanceConcentration
NaNO2 0.39 g/L MgSO4·7H20.025 g/L 
KH2PO4 0.1 g/L CaCl2·2H20.025 g/L 
NaHCO3 1.0 g/L Micronutrient 1 mL/L 

s-Fe0 (particle size of 2–3 mm) was purchased from Beijing Jinke Composites Co., Ltd in China. s-Fe0 has a density of about 2.30–2.78 g/cm3 and a bulk density of about 1.70–1.88 g/cm3. s-Fe0 mainly contains metallic iron (≥90%) with a total iron content of 96–97%. The s-Fe0 was pretreated before the experiments, and detailed information was presented in the study by Xie et al. (2021) .

Effect of s-Fe0 and carbon source on denitrification performance of iron-based autotrophic denitrification reactor

To determine the effects of s-Fe0 and carbon source on the denitrification performance of the reactor, four experimental systems were designed: (1) 75 mL inoculated sludge, labeled as the R0 system; (2) 75 mL inoculated sludge + 6.4 g s-Fe0, labeled as the R1 system; (3) 75 mL inoculated sludge + sodium acetate (102.6 mg/L), labeled as the R2 system; and (4) 75 mL inoculated sludge + 6.4 g s-Fe0 + sodium acetate (102.6 mg/L), labeled as the R3 system. When the reactor is started, the inoculated sludge needs to be washed to ensure the accuracy of the experiment. The inoculated sludge of 75 mL after domestication as described previously is taken, diluted to 150 mL with synthetic wastewater, and the supernatant is skimmed off after settling for 30 min. The dilution and settling process is repeated three times, and then centrifuged at 4,000 r/min for 5 min, dewatered, and put into the reactor. Sponge iron was added in a single dose, and sodium acetate was added to the synthetic wastewater and renewed periodically. All experiments were performed in amber glass flasks with a total volume of 250 and 160 mL of synthetic wastewater in the glass flasks. The reactor was operated at 30 °C and 160 rpm in a light-proof thermostatic shaker, and 80 mL of synthetic wastewater was replaced every 24 h (50% water exchange ratio). When replacing the synthetic wastewater, the reactor was removed and left for 30 min, then 80 mL of supernatant was poured out and replaced with an equal amount of synthetic wastewater according to the conditions, and the residual oxygen in the reactor was removed by N2 purging after reactor start-up and each water intake. The pH of the synthetic wastewater was adjusted to 7.0 ± 0.1 using NaOH (1.0 M) and HCl (1.0 M) if not otherwise specified. All steps were performed on a sterile operating table. During operation, the water was periodically taken out and filtered with 0.22 μm membrane and analyzed for NO3-N, NO2-N, NH4+-N, COD and TFe, and Fe(Ⅱ) concentrations and the pH of the effluent water. The TFe and Fe(Ⅱ) concentrations were measured every 9 days, and all other indicators were measured once every 3 days.

Effect of s-Fe0 dosing and C/N on denitrification performance of iron-based autotrophic denitrification system

Based on the aforementioned four reactors, to investigate the effects of s-Fe0 dosing and C/N on the denitrification performance of iron-based autotrophic denitrification system, experiments were conducted at different s-Fe0 dosings (0, 30, 40, 50 g/L) and C/N (0, 0.5, 1.0, 1.5). When investigating the effect of s-Fe0 dosage, all four reaction systems were injected with sodium acetate as the carbon source with C/N = 1 (102.6 mg/L), and the reactors were named as A, B, C, and D reactors from low to high according to the s-Fe0 dosage; when investigating the effect of C/N, all four reaction systems were injected with 40 g/L of s-Fe0, and the reactors were named as E, F, C (the reaction conditions were the same as C#, so they were not renamed here), and G# reactors from low to high according to C/N. Experiments were performed in 250 mL amber glass bottles, each containing 75 mL of activated sludge (dosed as shown in Section 2.2) and 160 mL of synthetic wastewater, and the reactor was operated at 30 °C and 160 rpm in a light-proof thermostatic shaker, as detailed in Section 2.2. The pH of the synthetic wastewater was adjusted to 7.0 ± 0.1 using NaOH (1.0 M) and HCl (1.0 M) if not otherwise specified. All steps were performed in a sterile operating table. After periodic sampling and filtration using 0.22 μm membrane, NO3-N, NO2-N, NH4+-N, COD, and TFe, Fe(Ⅱ) concentrations were analyzed, and the pH of the effluent water was detected. The analysis intervals are the same as presented in Section 2.2.

Analytical methods

The concentrations of NO3-N, NO2-N, and NH4+-N were measured with a 765 UV-Vis spectrophotometer (Shanghai, China) according to standard methods (Rice et al. 2012). The solution pH was monitored by a benchtop pH meter (PHS-3C, China). Considering the absence of organic nitrogen in the feed water, the concentration of TN in the solution was calculated from the sum of the concentrations of NO3-N, NO2N, and NH4+-N. VSS and SS values were measured according to Wang et al. (2014).

Effect of s-Fe0 and carbon source on denitrification performance of iron-based autotrophic denitrification system

The variation of each form of nitrogen with operating time and the average effluent concentration and average removal rate during the stabilization period for different reaction systems are shown in Figure 1.
Figure 1

The changes of each nitrogen index (as N) for each of the four bioreactor systems operated for 79 days under different conditions and the average effluent concentration and average removal rate of each group after entering the stabilization period: (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during stabilization period; and (f) average TN effluent concentration and removal rate during stabilization period. (initial solution pH = 7.0 ± 0.1).

Figure 1

The changes of each nitrogen index (as N) for each of the four bioreactor systems operated for 79 days under different conditions and the average effluent concentration and average removal rate of each group after entering the stabilization period: (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during stabilization period; and (f) average TN effluent concentration and removal rate during stabilization period. (initial solution pH = 7.0 ± 0.1).

Close modal

As shown in Figure 1, the nitrogen in each form in the reaction system of the four groups showed dramatic fluctuations at the beginning of the reaction. The effluent index of each form of nitrogen in the R3 system reached stability at about 31 cycles of operation, while the remaining three groups entered the stability period only at about 43 cycles of operation, which proved that the simultaneous intervention of sponge iron and carbon source promoted the start-up of the reaction system, while the addition of sponge iron or carbon source alone had no significant effect on the start-up speed of the reaction system. In R0, there was a certain degree of ammonia nitrogen production in the reactor in the first 15 days. This may be due to the lack of organic matter leading to a large number of microbial deaths, and the decomposition of proteins during the disintegration process leading to the production of NH4+-N (Chamchoi & Nitisoravut 2007). Disintegrating microorganisms can provide organic carbon sources for specialized heterotrophic and mixotrophic microorganisms to promote their growth and development and function. In contrast, the death of a large number of denitrifying bacteria directly led to a decrease in the effectiveness of the reactor for nitrite removal, so that the effluent concentration of nitrite increased from 0 to 76.31 mg/L within 8 days. Due to the weakening of microbial competition for nitrite, more nitrite is oxidized by Fe(Ⅲ) or residual oxygen molecules in the wastewater to produce nitrate. This process may be facilitated by some nitrifying bacteria, as the functional microorganisms present in the inoculated sludge are diverse. The R0 system showed a poor removal effect on nitrite, with an average TN effluent concentration of 73.55 mg/L and an average TN removal rate of only 8.21% during the stabilization period.

In R1, the addition of sponge iron allows for more ammonia nitrogen production at the beginning of reactor start-up, as s-Fe0 itself is reduced to ammonia nitrogen with nitrite. In addition, toxicity from an iron-rich environment may contribute to partial cell lysis and also to elevated NH4+-N concentrations in solution. This phenomenon occurs only in the pre-reactor stage, because as the reaction proceeds, the sponge iron will continue to passivate gradually weakening the chemical reaction and the dissolution of iron ions, which is verified by the change in the concentration of Fe(Ⅱ) (Figure 2(d)). The average TN effluent concentration and removal rate after R1 stabilization were 68.69 mg/L and 14.58%, respectively, and the reaction performance was slightly better than that of R0. This may be due to the fact that sponge iron promotes biological denitrification in the following aspects: (1) sponge iron can participate in electron transfer as an electron donor for iron autotrophic denitrification, improving the efficiency of electron generation, transfer, and consumption in the process of biological denitrification (Wang et al. 2020a); (2) sponge iron can significantly reduce the redox potential of denitrifying sludge, creating a reducing environment suitable for the growth of certain specialized anaerobic bacteria including denitrifying bacteria (Zhang et al. 2014); (3) the many loose micropores on the surface of sponge iron provide a good habitat for denitrifying bacteria; and (4) iron (Fe) is a crucial element in cytochromes found in microbial denitrifying enzymes, and the use of sponge iron can increase the relative abundance of denitrifying bacteria and improve denitrification performance (Sun et al. 2019); (5) for the enzymatic iron oxidation denitrification process, in addition to Fe(II), hydrogen produced by the dissolution of Fe(0) can also serve as a reasonably good electron donor for denitrifying bacteria (Jiang et al. 2020). It is noteworthy that the conversion of nitrite to nitrate was inhibited by the addition of sponge iron after 15 days and was not accompanied by the production of ammonia nitrogen. This is because the presence of sponge iron inhibits the conversion of nitrite to nitrate. On the one hand, sponge iron (fresh) itself will reduce to nitrite. On the other hand, as a water deoxidizer, its high porosity, specific surface area, and activity make sponge iron react easily with oxygen in water to generate Fe3O3, so as to achieve the purpose of deoxidizing and inhibit the solution of residual oxygen on the conversion of nitrite to nitrate. During the conversion of nitrite to ammonia nitrogen, fresh sponge iron acts as an electron donor. However, as the reaction progresses, a passivation layer inevitably forms on the sponge iron surface, significantly reducing its reaction activity. This limits the conversion of nitrite to ammonia nitrogen. A similar phenomenon was observed in R3 due to the addition of sponge iron. Abiotic reactions that may result from the addition of sponge iron under anoxic conditions include the following: (1) Fe(0) is oxidized and dissolved by water, releasing Fe(II), hydroxide, and hydrogen; (2) direct reduction of nitrite to ammonium by Fe(0), accompanied by proton depletion and formation of Fe(II); and (3) nitrite oxidizes Fe(II) to Fe(III), producing other reduced forms of inorganic nitrogen, such as nitric oxide and nitrous oxide, in addition to reduced proton concentrations (Tratnyek et al. 2003; Liang et al. 2008; Klueglein et al. 2014).
Figure 2

Changes in the indicators of each of the four bioreactor systems operating for 79 days under different conditions: (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ) (initial solution pH = 7.0 ± 0.1).

Figure 2

Changes in the indicators of each of the four bioreactor systems operating for 79 days under different conditions: (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ) (initial solution pH = 7.0 ± 0.1).

Close modal

The changes in ammonia nitrogen concentration in R2 and R0 were extremely similar (Figure 1(c)), indicating that the carbon source dosing did not significantly inhibit the microbial disintegration process. Therefore, the role of the carbon source may mainly lie in providing nutrients to promote more microbial colonization. The lower nitrate concentration observed in R2 compared to R0 indicates that the carbon source injection inhibited the nitrate production in the reactor, probably due to the enhanced activity of denitrifying bacteria supported by the carbon source, thus promoting their competition for nitrite, although the effect was much less than that of the addition of sponge iron. The average TN effluent concentration and removal rate after R2 stabilization were 56.50 mg/L and 29.50%, respectively. The addition of carbon source increased the average TN removal rate by 21.29%, while the addition of s-Fe0 only increased it by 6.37%, which indicates that carbon source plays a more critical role in promoting biological denitrification by promoting the growth of both heterotrophic denitrifying bacteria and iron-based autotrophic denitrifying bacteria. Microorganisms are the main contributors to denitrification in the reaction system. However, the respective contributions of heterotrophic denitrifying bacteria and iron-based autotrophic denitrifying bacteria to nitrogen removal in the reaction system need further characterization and analysis.

Among the four groups of reaction systems, R3 had the fastest start-up speed and the highest TN removal efficiency, and was the best reaction treatment system. The average TN effluent concentration and removal rate of R3 stabilized effluent were 41.94 mg/L and 47.87%, respectively. The addition of sponge iron or carbon source is beneficial to increase species diversity, enrich the denitrification pathway, and improve denitrification efficiency and stability. Compared with R1, R3 had lower ammonia nitrogen effluent, which might be due to the addition of carbon source to enhance the competition of microorganisms for nitrite and inhibit the conversion of nitrite to ammonia nitrogen to some extent. The average TN removal rate of R3 is higher than the sum of R1 and R2, and therefore, R3 cannot be simply regarded as a combination of R1 and R2 systems. There is a synergistic effect between sponge iron and carbon source, which will further enhance the overall nitrogen removal efficiency of the system. This is mainly reflected in the following aspects: (1) the carbon source can improve the microbial activity and enhance its microbial-induced corrosion (MIC) effect on sponge iron, which in turn provides more Fe(Ⅱ) as an electron donor to participate in the denitrification process. (2) The presence of an organic carbon source in a low carbon-to-nitrogen ratio environment enhances the microbial use of Fe(Ⅱ) and reduces cellular crusting due to Fe autotrophic denitrification to some extent. In conclusion, the presence of organic carbon sources can support the basic metabolic activity of microorganisms and enhance the process of iron-based autotrophic denitrification, thereby avoiding the reliance of the denitrification process on a single electron donor.

The variation of COD, pH, and mixed solution TFe and Fe(Ⅱ) concentrations of effluent from different reaction systems with running time is shown in Figure 2.

During the first 6 days after reactor start-up, COD effluents exceeding the influent concentration were observed in each reaction system, and these CODs were thought to be formed due to microbial death and disintegration, which is consistent with the findings observed in the variation of ammonia nitrogen concentrations. The COD concentration changes of R0 and R2 were close to each other, which further confirmed that there was no significant effect of carbon source on the microbial death disintegration process. Uninterrupted COD production was observed throughout the operation of R0 and R1 systems, which indicated that the reaction system had been accompanied by microbial death due to nutrient deficiency or iron toxicity, which might be an important reason for the low denitrification efficiency of both systems. The R1 system, which did not inject a carbon source, instead exhibited the highest COD effluent at the beginning of start-up, suggesting that Fe s-Fe0 can have a strong degree of toxic effect on microorganisms at the beginning of input to the reactor. Very little COD effluent was observed in the R2 and R3 systems with the carbon source, which indicates that the COD was well utilized in these two systems and the addition of a moderate amount of carbon source did not cause secondary pollution in this system.

The addition of both s-Fe0 and carbon source led to the increase in pH in the effluent of the reaction system. On the one hand, the chemical reaction of s-Fe0 with nitrite produced a large amount of alkalinity, and on the other hand, the addition of carbon source led to the increase of biological denitrification, which produced more alkalinity. It is clear that s-Fe0 dosing contributes more to pH enhancement, either by Fe(II) or hydrogen as an electron donor, and this nitrite respiration pathway likely requires protons to be involved as reactants (Kopf et al. 2013; Bruce 2020). The elevation of pH was observed throughout the experiment, which suggests that s-Fe0 continues to play a role in the reaction system, manifesting itself in the early stages of the reaction system initiation as a chemical reaction between s-Fe0 and nitrite to produce ammonia nitrogen, whereas in the later stages of the experiment, passivated s-Fe0 does not react directly with nitrite, but rather reacts with nitrite to produce N2O through the solubilization of Fe(II).

Higher TFe concentrations were observed in the reaction systems of R1 and R3 with the addition of sponge iron, suggesting that sponge iron would be dissolved into the wastewater in the ionic state. In contrast, comparing R1 and R3, it was found that the addition of carbon source promoted more dissolution of sponge iron, probably because the carbon source ensured the microbial activity and thus promoted the microbially induced corrosion (MIC) of sponge iron, which supports the synergistic effect of sponge iron and carbon source mentioned in the previous section. The large amount of Fe(Ⅱ) production in R0 and R2 during 4–25 days, when a large amount of nitrate nitrogen production was observed in both reaction systems at the same time, may be due to the presence of residual dissolved oxygen in the system, leading to the reaction of nitrite with Fe(Ⅲ) to form Fe(Ⅱ) and nitrate, which further confirms the previous speculation. However, whether this process is chemical or microbially driven needs further demonstration, nitrifying bacteria may also be present in the system.

Effect of s-Fe0 dosing on the denitrification performance of iron-based autotrophic denitrification system

The variation of each form of nitrogen with operating time and the average effluent concentration and average removal rate during the stabilization period for different s-Fe0 dosages (0, 30, 40, and 50 g/L) of the reaction system at C/N = 1 are shown in Figure 3.
Figure 3

The changes of each nitrogen index (as N) and the average effluent concentration and average removal rate of each group after entering the stabilization period for each of the four bioreactor systems operated for 79 days at different s-Fe0 dosages (0, 30, 40, 50 g/L): (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during stabilization period; (f) average TN effluent concentration and removal rate during stabilization period. (Initial solution pH = 7.0 ± 0.1, and carbon sources were added to all reaction systems at a ratio of C/N = 1.)

Figure 3

The changes of each nitrogen index (as N) and the average effluent concentration and average removal rate of each group after entering the stabilization period for each of the four bioreactor systems operated for 79 days at different s-Fe0 dosages (0, 30, 40, 50 g/L): (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during stabilization period; (f) average TN effluent concentration and removal rate during stabilization period. (Initial solution pH = 7.0 ± 0.1, and carbon sources were added to all reaction systems at a ratio of C/N = 1.)

Close modal

As can be seen from Figure 3, the reactor effluent indexes fluctuated greatly when the sponge iron dosage was 0 g/L. Drastic fluctuations in nitrite effluent were observed from 6 to 43 days. This may be due to the unstable conversion of nitrite to nitrate prompted by residual oxygen molecules in an uncritical anaerobic environment, and the reactor reached stable effluent after 43 days of operation, which was regarded as a successful start-up. The stable average effluent concentration and average removal rate of TN were 56.50 mg/L and 29.50%, respectively, at the sponge iron dosage of 0 g/L. After 31 operation cycles, all indicators in the reaction system with sponge iron dosage reached a more stable effluent, and the sponge iron dosage could stabilize the denitrification effect of the reaction system and significantly accelerate the start-up speed, but the three sponge iron dosages under the experimental conditions did not have any effect on the start-up speed of the reaction system. However, under the experimental conditions, the dosage of the three types of sponge iron did not have a significant effect on the start-up speed of the reaction system. The reaction system showed the best denitrification effect when the iron sponge was added at 30 g/L. The average TN effluent concentration and the average removal rate during the stabilization period were 38.05 mg/L and 52.66%, respectively, while the worst denitrification effect was 40 g/L. The average TN effluent concentration and the average removal rate during the stabilization period were 41.94 mg/L and 47.9%, respectively. The difference between the steady-state TN removal rate of 41.94 mg/L and 47.84% was less than 5%, which indicated that the amount of iron sponge dosing in the experimental range of the reaction system might not be a decisive factor affecting the denitrification effect. The removal effect of nitrite and TN in the reactor with sponge iron was significantly better than that in the reactor without sponge iron, which proved the promotion of sponge iron for the denitrification effect of the reaction system.

At the sponge iron dosage of 0 g/L, a much smaller amount of ammonia nitrogen production was observed in the reaction system because there was no direct reaction between zero-valent iron and nitrite to produce ammonia nitrogen, and the only source of ammonia nitrogen at this time was protein disintegration during microbial death, and after about 15 days, the microbial disintegration process ended and ammonia nitrogen production was almost no longer observed in the reactor. In contrast, higher ammonia nitrogen concentrations were observed in all the other three reaction systems, probably due to the large conversion of nitrite to ammonia nitrogen by the fresh sponge iron. The higher the amount of sponge iron dosing, the higher the concentration of ammonia nitrogen, indicating that the aforementioned process was also enhanced. In addition, a large amount of nitrate production was observed only at 0 g/L of sponge iron dosage, indicating that all three sponge iron dosages under the experimental conditions could effectively ensure the anaerobic environment of the system and inhibit the conversion of nitrite to nitrate.

The variation of COD, pH, and mixed solution TFe and Fe(Ⅱ) concentrations with running time for each reaction system at different s-Fe0 dosages (0, 30, 40, and 50 g/L) for C/N = 1 is shown in Figure 4.
Figure 4

Changes in the indexes of the four bioreactor systems operating for 79 days at different s-Fe0 dosing levels (0, 30, 40, and 50 g/L): (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ). (Initial solution pH = 7.0 ± 0.1 and carbon source was added to all reactors at a ratio of C/N = 1.)

Figure 4

Changes in the indexes of the four bioreactor systems operating for 79 days at different s-Fe0 dosing levels (0, 30, 40, and 50 g/L): (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ). (Initial solution pH = 7.0 ± 0.1 and carbon source was added to all reactors at a ratio of C/N = 1.)

Close modal

There was almost no COD residue in each reactor, which proved the role of heterotrophic denitrification in the experimental system. The COD concentration in the pre-operation period of the reaction system was slightly higher than the other three groups of reaction systems when s-Fe0 was injected at 0 g/L. This may be because s-Fe0 could create an environment more favorable to the microbial growth, which alleviated microbial death and disintegration to a certain extent and promoted the microbial uptake of carbon sources and the uptake and utilization of the carbon source by microorganisms. There was almost no difference in COD concentrations observed in the reaction system with sponge iron dosing, indicating that the three dosing amounts under experimental conditions did not significantly affect microbial mortality and carbon source utilization. After the eighth day of operation, almost no COD effluent was observed in the four reaction systems, indicating that the carbon source could be effectively utilized by microorganisms under the experimental conditions regardless of the s-Fe0 dosage.

There was no significant relationship between effluent pH and s-Fe0 dosing, and the effluent pH of the reactor with s-Fe0 dosing of 0 g/L was significantly lower than that of the other reactors. This may be because on the one hand, the chemical denitrification of sponge iron itself with nitrite is the alkali-producing process, and on the other hand, the iron-type denitrification reaction occurring in this reactor may lead to an increase in alkalinity.

As the amount of sponge iron added increases, the concentration of TFe in the reaction system increases, and higher sponge iron addition is accompanied by more dissolution of iron ions. In the reactor without sponge iron, the concentration of TFe gradually decreases, because with the discharge of water, there must be a loss of ferrous ions and no continuous dissolution of sponge iron to replenish ferrous ions. The ferrous ion concentration gradually decreased and remained low as the experiment progressed, probably because it was gradually consumed as an electron donor in the reaction system.

Effect of nitrogen removal performance of C/N iron-based autotrophic denitrification system

The variation of each form of nitrogen with operating time and the average effluent concentration and average removal rate during the stabilization period of the reaction system at different C/N (0, 0.5, 1.0, and 1.5) for the s-Fe0 dosage of 40 g/L are shown in Figure 5.
Figure 5

The changes of each nitrogen index (as N) and the average effluent concentration and average removal rate of each group after entering the stabilization period at different C/N (0, 0.5, 1, 1.5) for each of the four bioreactor systems operating for 79 days: (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during the stabilization period; (f) average effluent concentration and removal rate of TN during stabilization period. (The initial solution pH = 7.0 ± 0.1, and the s-Fe0 dosage was 40 g/L in all reaction systems.)

Figure 5

The changes of each nitrogen index (as N) and the average effluent concentration and average removal rate of each group after entering the stabilization period at different C/N (0, 0.5, 1, 1.5) for each of the four bioreactor systems operating for 79 days: (a) NO2-N concentration and its removal rate; (b) TN concentration and its removal rate; (c) NH4+-N concentration; (d) NO3-N concentration; (e) average effluent concentration and removal rate of NO2-N during the stabilization period; (f) average effluent concentration and removal rate of TN during stabilization period. (The initial solution pH = 7.0 ± 0.1, and the s-Fe0 dosage was 40 g/L in all reaction systems.)

Close modal

As the C/N ratio increased from 0 to 1.5, the stable average effluent concentration of nitrite from the reactor gradually decreased from 67.42 to 28.13 mg/L, and the stable average removal rate gradually increased from 16.61 to 64.98%; the stable average effluent concentration of TN gradually decreased from 68.69 to 28.99 mg/L, and the stable average removal rate gradually increased from 14.58 to 63.92%. When the C/N ratio = 0, it took 43 days to stabilize the reactor, and it took about 31 days when the C/N ratio was 0.5 and 1. When the C/N ratio was increased to 1.5, the nitrogen indexes of the reactor effluent were stabilized after 25 cycles of operation. The addition of carbon source could significantly improve the removal efficiency and stability of the reactor and accelerate the start-up of the reactor, and the aforementioned effect was enhanced gradually with the increase of the C/N ratio. The carbon source may play two important roles in the reaction system: first, as a direct electron donor for denitrification by heterotrophic denitrifying bacteria, and second, as a nutrient for iron autotrophic denitrifying bacteria to be absorbed and used to maintain their high denitrification performance. In addition, the high nitrate fluctuation in the reaction system at C/N = 0, even if sponge iron was added to the system, suggests that the carbon source also played a role in inhibiting the conversion of nitrite to nitrate, probably because the addition of the carbon source increased the activity of denitrifying bacteria and thus enhanced the competition of denitrification for nitrite.

The variation of COD, pH, and mixed solution TFe and Fe(Ⅱ) concentrations with running time for each reaction system at different C/N ratios (0, 0.5, 1.0, 1.5) for s-Fe0 dosage of 40 g/L is shown in Figure 4.

Under experimental conditions, the carbon source was efficiently utilized at all C/N, avoiding secondary contamination (Figure 6(a)). Small fluctuations in COD concentrations were observed at lower C/N ratio (0 and 0.5), and these CODs are thought to be produced by nutrient deficiencies leading to microbial mortality.
Figure 6

Changes in the indicators of each of the four bioreactor systems operated for 79 days at different C/N (0, 0.5, 1, 1.5): (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ). (The initial solution pH = 7.0 ± 0.1, and the s-Fe0 dosage was 40 g/L in all reaction systems.)

Figure 6

Changes in the indicators of each of the four bioreactor systems operated for 79 days at different C/N (0, 0.5, 1, 1.5): (a) COD, (b) pH, (c) TFe, and (d) Fe(Ⅱ). (The initial solution pH = 7.0 ± 0.1, and the s-Fe0 dosage was 40 g/L in all reaction systems.)

Close modal

The effluent pH increased with the increasing C/N ratio, which may be caused by more alkalinity produced by the denitrification process at higher C/N ratios. The Fe(Ⅱ) content in the solution gradually decreased due to the occurrence of iron-based autotrophic denitrification reaction. The higher Fe(Ⅱ) concentration observed in the reaction system with a higher C/N ratio may be due to the fact that higher carbon source concentration increases microbial activity, which in turn enhances MIC to promote Fe(Ⅱ) leaching from sponge iron, which fully demonstrates the synergistic effect of iron-based autotrophic denitrifying bacteria and heterotrophic bacteria, corroborating the previous conclusion.

The natural rate of Fe(II) dissolution from sponge iron is uncontrollable, and the dissolution rate tends to gradually decrease with the accumulation of iron minerals on the sponge iron surface, resulting in reaction stagnation. It is suggested that the DC electric field can be coupled with the iron-based autotrophic denitrification system with sponge iron injection to control the dissolution rate of Fe(II) by controlling the current size, in order to weaken the Fe(II) dissolution in the early stage of the experiment to weaken its biotoxicity, and to enhance the Fe(II) dissolution in the late stage of the experiment to meet the electron donor demand of denitrification.

Engineering applications and research directions

The sponge iron-driven iron-based autotrophic denitrification process can further reduce the concentration of nitrogen in the tail water on the basis of the traditional nitrification denitrification mainstream process, which not only can reduce the carbon input in the denitrification process but also helps to ensure the stable iron-based autotrophic denitrification activity in the effluent low C/N environment. Among them, the inoculated sludge can be selected from anaerobically digested sludge, which can adapt to the iron-rich environment and shorten the start-up time of the iron-based autotrophic denitrification process. Second, the iron-rich sludge produced during the long-term iron-based autotrophic denitrification process can be directly discharged into the anaerobic digester to improve the methane yield.

In order to improve and scale up iron-based autotrophic denitrification technology for practical applications in water treatment and pollution control, the following studies are still needed: (1) regeneration of ferrous iron (avoiding continuous dosing of ferrous iron sources); (2) determination of reactor configurations suitable for iron-based autotrophic denitrification (sponge iron may not only act as an indirect electron donor in iron-based autotrophic denitrification but also the direct attachment of microorganisms to the surface of the sponge iron may be more conducive to electron transfer (sponge iron acting as a biocarrier) than suspended sludge); (3) determination of electron transfer mechanisms (limited free ferrous ions during sponge iron-driven iron-based autotrophic denitrification may not be able to support large amounts of nitrogen removal, thus necessitating further exploration of the feasibility of extracellular electron transfer); (4) feasibility study of solid-phase electron shuttles for nitrogen removal performance enhancement (based on (3), assuming that extracellular electron transfer dominates the exchange of information in the iron-based autotrophic denitrification process driven by sponge iron, solid-phase electron shuttles, such as biochar, can be considered to enhance nitrogen removal, wherein biochar can be prevented from being lost in large quantities over the course of its use).

In this study, the nitrogen removal effect of iron-based autotrophic denitrification process driven by sponge iron for nitrite removal under anaerobic conditions was investigated, and the roles of sponge iron and carbon source in this reaction system were elucidated. This experiment was conducted for 79 days, optimum parameters for TN removal in the iron-based autotrophic denitrification system were determined based on the s-Fe0 dosage of 30 g/L and C/N = 1.5. The influence of two factors on the denitrification effect in the iron-based autotrophic denitrification system was ranked as C/N > s-Fe0 dosage. Both sponge iron and carbon source could effectively promote the removal of nitrite and inhibit the conversion of nitrite to nitrate in the reaction system. The nitrogen removal efficiency of the combined system with both sponge iron and carbon source was higher than that of the single system with sponge iron or carbon source. Sponge iron provides Fe(Ⅱ) while ensuring the anaerobic environment of the reaction system, and the numerous loose micropores on the surface can also provide a good growth environment for microorganisms. In addition, Fe serves as an important component of cytochromes in microbial denitrifying enzymes, and sponge iron dosing helped to increase the relative abundance of denitrifying bacteria; the carbon source played an important role in maintaining microbial growth and activity and also promoted Fe(Ⅱ) leaching by enhancing the MIC process to achieve a synergistic effect. These results can help us to gain more insight into the global iron and nitrogen cycle and provide more theoretical basis for further control of nitrogen pollution in water bodies.

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

The authors declare there is no conflict.

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