Fe(III) bio-reduction provides a prospect of applying the iron redox cycle to nitrate remediation in the aquatic environment. The objective of this study was to realize multiple nitrate removals in the system containing Shewanella oneidensis MR-1 (S. oneidensis MR-1) and ferrihydrite or magnetite. The results showed that with three periods of 30 mg·L−1 NO3-N addition, all nitrate reduction was completed within 170 h. In the first period (0–30 h) of nitrate addition, the main contribution of nitrate removal was due to the biological reduction process by S. oneidensis MR-1, accompanied by the reduction of Fe(III). During the second (45–90 h) and third periods (100–170 h) of nitrate addition, oxidation of biogenic Fe(II) coupled with the reduction of nitrate took place. This redox reaction resulted in the production of gaseous nitrogen of 47.33% and 16.8% for ferrihydrite/S. oneidensis MR-1 and magnetite/S. oneidensis MR-1 systems, respectively. In addition, nitrite, as an intermediate product, accumulated and negatively affected nitrate removal after the third addition of nitrate. By comparing the patterns of X-ray diffraction of the iron minerals before and after the bio-reduction, it was found that ferrihydrite was transformed into magnetite, while magnetite kept its original crystal form.

  • Low-concentration nitrate did not inhibit the bio-reduction of Fe(III).

  • Nitrate was removed continuously in a microbial Fe redox cycle.

  • Abiotic reduction with biogenic Fe(II) was the sole process to remove TN.

  • Nitrite accumulated might have a negative effect on nitrate removal.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Nitrogen is an indispensable nutrient in the process of human survival. However, extensive application of nitrogen fertilizers, leakage of landfill leachate and septic tanks can lead to the accumulation of nitrate in surface water and groundwater, causing a series of environmental and health issues (Howarth 2004). Remediation of nitrate contaminated water becomes a significant environmental challenge worldwide (Wu 2020). Among the numerous remediation techniques, bioremediation is a process of removing nitrate through microbial metabolisms, commonly known as denitrification. However, the lack of available organic carbon is recognized as the largest hindrance to denitrification (Cao & Zhou 2019). By comparison, chemical reductions such as those facilitated with zero-valent iron (Fe0) have been widely applied due to the strong reduction ability (Park et al. 2009). However, a previous study found that ammonium (NH4+) was the end-product of the nitrate reduction by Fe0 (Suzuki et al. 2012), making it an incomplete nitrogen removal process. It there is an urgent need to develop an environment friendly, sustainable and effective method for removing nitrate.

Fe(II), which is both reductive and oxidative, plays an important role in the removal of pollutants, and is easily oxidized (Melton et al. 2014). Moreover, in nature, iron mainly exists in the form of different iron minerals, among which ferrihydrite and magnetite are the typical representatives of weakly crystalline and strongly crystalline iron minerals, respectively. The application of dissimilatory iron reduction bacteria (DIRB) can transform structured Fe(III) into Fe(II) and improve the utilization rate of iron minerals (Kappler et al. 2021). Knowledge of microbial-mediated Fe(III) reduction process has grown over the past few decades, and is mostly a process of reducing Fe(III) to Fe(II) coupled with organic compound oxidation under anoxic conditions, driven by DIRB (Pan et al. 2019). During dissimilatory iron reduction, DIRB solubilise Fe(III) oxides as Fe(II), known as biogenic Fe(II) (Roden & Urrutia 2002). With the reduction of Fe(II), sulfide, heavy metals and organic pollutants can be reduced to restore water (Melton et al. 2014). They can also be used to form vivianite combined with phosphate and to generate high economic profits (Wang et al. 2018). Therefore, the iron bio-reduction process has promising potential for nitrate contaminated water remediation.

Among these iron-reducing bacteria, Geobacter sp. and Shewanella sp. have been widely used (Lovley et al. 2004). The iron bio-reduction process is relatively slow, so it is necessary to add electron shuttles to accelerate the occurrence of the iron bio-reduction process (Zhou et al. 2016; Yang et al. 2019). It was also found that S. oneidensis MR-1 bacteria themselves could secrete electron shuttles (Marsili et al. 2008; Peretyazhko et al. 2010). S. oneidensis MR-1 could use Fe(III) and nitrate as electron acceptors, while high-concentration nitrate inhibited the growth of S. oneidensis MR-1 (Myers & Nealson 1990). Li and his colleagues also found that high concentrations of nitrate as electron acceptors could inhibit the bio-reduction of iron minerals (Li et al. 2015). The DIRB/nitrate-dependent Fe(II)-oxidizing coculture system could undergo three redox cycles during a period of 220 days with nontronite NAu-2 (total Fe content of NAu-2 is 24%, of which 99.4% is Fe(III)) and biogenic Fe(II) serving as the electron acceptor and donor, respectively (Zhao et al. 2015). This result indicated that the biotic process achieved multiple removal cycles over an extended period of time. Our previous work showed that microbial-mediated multiple iron redox cycles played an important role in nitrite removal with a low nitrite concentration of 30 mg·L−1 (Lu et al. 2017). However, the above-mentioned studies mainly focused on nitrite through denitrification by microbial-mediated iron reduction (Lu et al. 2020), while few studies have been carried out on nitrate. Additionally, in nitrate-rich systems, the intermediate product nitrite would be produced, which inhibits microbial activity and negatively affects the removal of nitrate (Cooper et al. 2003). The removal of nitrite by S. oneidensis MR-1-mediated iron mineral reduction is in the early stages of being studied. Within 180 h, both the systems of S. oneidensis MR-1 with ferrihydrite and S. oneidensis MR-1 with magnetite could complete at least four and six cycles, respectively (Lu et al. 2017). Therefore, it would be expected to remove nitrate in an iron minerals system containing iron-reducing bacteria based on the iron recycling process. However, nitrite can react with both biogenic Fe(II) and non-biological Fe2+, but nitrate can react with biogenic Fe(II) (Melton et al. 2014; Kappler et al. 2021). The questions of nitrate removal by iron reduction processes, then, are: (1) whether the mechanism of nitrate removal through iron minerals reduction mediated by S. oneidensis MR-1 is the same as that of nitrite removal, and (2) how many iron cycles can be achieved within 180 h. Understanding the mechanisms of nitrate removal would also be helpful in improving the application prospect of denitrification by iron minerals. Therefore, it is important to investigate the role of biogenic Fe(II) in nitrate removal. The objectives of this study, were to investigate: (1) the biological and abiotic processes of nitrate removal, (2) the nitrogen transformation pathway and the iron minerals transformation, and (3) the mechanisms underlying nitrate and nitrite removal in the iron mineral reduction process mediated by S. oneidensis MR-1.

Minerals and cultures preparation

In this study, iron oxide ferrihydrite or magnetite was used for nitrate removal. Briefly, ferrihydrite was synthesized by neutralization of the FeCl3·6H2O (n2+Fe = 0.4 M) solution with NaOH (nOH = 1 M), for which the final pH was 7.0, and the above solution was centrifuged (4,000 rpm,10 min), then the supernatant was removed and washing with deionized water was repeated two or three times (Lovley & Phillips 1986). Magnetite was prepared by the co-precipitation method of FeCl2·4H2O (n2+Fe = 0.05 M) and FeCl3·6H2O (n2+Fe = 0.1 M), and dissolved under vigorous stirring at 80°C under a nitrogen atmosphere. After that, NH4OH (25 wt.%) was added and continuously stirred for 30 min, then centrifuged (10,000 rpm, 10 min) and washed three or four times with deionized water (Hu et al. 2014).

S. oneidensis MR-1, as the model bacteria for DIRB, was provided by the Marine Culture Collection of China (MCCC). The cells were incubated in Luria-Bertani medium (5 g·L−1 NaCl, 10 g·L−1 tryptone, 5 g·L−1 yeast extract) at 30 ± 0.5 °C in a dark, constant temperature, incubator shaker. Bacteria were collected by centrifugation at 10,000 rpm for 10 min when the cell growth reached the mid-log phase (around 18 h). After washing with 0.9% NaCl three times, and diluted with 0.9% NaCl, a cell suspension was obtained (Xu et al. 2014). The final concentration of S. oneidensis MR-1 bacteria was about 5 × 107 cells·mL−1 in all experimental systems in this study. The procedure for the experimental process is shown in Figure 1. A series of batch experiments was set up as follows with iron minerals and S. oneidensis MR-1 bacteria.

Figure 1

Flow diagram of experiment.

Figure 1

Flow diagram of experiment.

Close modal

Batch experiments

All the experimental systems were established in 100 mL anaerobic serum bottles, and the volume of the final culture system was 80 mL. Lactate (C3H5O3Na) 50 mM was added as the electron donor, ferrihydrite or magnetite (2,500 mg·L−1 total Fe) was added as the electron acceptor. In addition, sodium anthraquinone-2,6-disulfonate (AQDS,100 μM) was added as the electron shuttle to enhance the effects on biotic reduction. For studying the removal of nitrate of different concentrations, the concentration of NO3-N was controlled at 30, 70, 140, and 280 mg·L−1. In the iron minerals-only system, the volume of the system was adjusted to 80 mL with a mineral medium (5.85 g·L−1 NaCl, 0.67 g·L−1 NaH2PO4·2H2O, 0.3 g·L−1 NaOH, 0.097 g·L−1 KCl and 1 mL·L−1 of trace elements solution (Lu et al. 2017)). In the culture system for iron minerals and microorganisms, the culture system was adjusted to 40 mL with the same mineral medium, before 40 mL of cell suspension was added. All systems were then capped with a rubber stopper after a 5-min anaerobic treatment (99.99% He gas), then incubated in a thermostatic shaker (100 rpm) and maintained at 30 ± 0.5 °C in the dark. The final pH was 6.8. In the iron cycle experiment, the method of system establishment was the same as above. The timings of the 30 mg·L−1 nitrate addition were 0, 45 and 100 h. The first and second additions of nitrate were termed the first period and the operational period, respectively. All materials except iron minerals were autoclaved at 121 °C for 30 min before use. Each treatment in this study had three replicates.

Analytical methods

The reaction mixtures were sampled by syringes and filtered through 0.22 μm PTFE (polytetrafluoroethylene) filters to remove iron oxides and organic components. Standard methods were used to measure NO2-N, NO3-N and NH4+-N (Peretyazhko et al. 2010; Lu et al. 2017). The biogenic Fe(II) produced by the iron reduction process contained various forms, not only dissolved, but also colloidal, particle, adsorption, etc. Different types of Fe(II) particle size were present. In the measurement for Fe(II), the filter head was used to filter the organic matter to not affect the measurement of Fe(II). The extraction process could decompose Fe(II) with large particle size into small Fe(II), reducing the measurement error of Fe(II) concentration (Peretyazhko et al. 2010), as determined by the ferrozine method. Here, a 1 mL aliquot was extracted for 1.0 h in 6 M HCl to dissolve the insoluble iron oxide complete.

For gaseous nitrogen measurements, the air from the headspace of the serum bottle was detected by gas chromatography (GC 2030 plus, Shimadzu, Japan) with an SH-Rtx-5 column (30 m × 0.25 mm × 0.25 μm) and an FID detector. The operation conditions included an oven temperature of 60 °C and an FID temperature of 330 °C. The external standard method was used to quantify gas concentrations. The peak was CO2 in 2.40 min, N2O in 3 min and NO2 in 1.40 min. The change of ferrihydrite or magnetite during redox cycling of nitrate was analyzed from their X-ray diffraction (XRD) patterns, which were recorded on a Rigaku D/max RBX X-ray diffractometer with Cu-Kα radiation (λ = 0.154 nm) and a scanning rate of 8°·min−1 in the 2θ range of 5–80°. After each experimental run, the serum bottles were allowed to settle for 10 min. The supernatant was then gently poured into a 50 mL sterile centrifuge tube for high-speed centrifugation (10,000 rpm, 10 min). The supernatant was discarded and the bacterial precipitate was collected. The activity of the cells was then observed through a laser confocal microscope (Olympus FV1200).

The data were statistically analyzed using Origin (version 9.0, Massachusetts, OriginLab, USA) and results were expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) with Duncan test was performed at the significance level of p < 0.05 using SPSS (version 18.0, Chicago, Illinois, USA). The removal rate of nitrate and the proportion of different kinds of nitrogen compounds in the transformation products of nitrate are shown in Equations (1) and (2), respectively.
formula
formula
in which, C0 is the initial concentration of nitrate (mg·L−1); C is the concentration of nitrate in the reaction process (mg·L−1); and Ct is the concentration of different nitrogen compounds (mg·L−1).

Comparison of nitrate removal with or without iron oxides

No significant nitrate reduction was observed in the control systems only with ferrihydrite or magnetite (Figure 2(a) and 2(b)), illustrating that iron minerals had no reductive activity for nitrate (Zhu et al. 2014). When S. oneidensis MR-1 bacteria incubated with nitrate and lactate, 90% of NO3-N was reduced in 12 h with initial concentrations of 30 mg·L−1 and 70 mg·L−1 (Figure 2(c)), indicating that nitrate can be used as electron acceptor for the growth of S. oneidensis MR-1 bacteria (Lovley et al. 2004). Inhibition of microbial activities could be detected with the increase in NO3-N concentration (Figure 2(c)). This was likely to be because nitrate could enter the periplasmic nitrate reductase system (NAP) located in S. oneidensis MR-1 cells and reduced nitrate to nitrite, which was then reduced to ammonium by the nitrite reductase system (NRF) (Cruz-Garcia et al. 2007; Simpson et al. 2010). During this process, nitrite accumulated, and the toxicity of nitrite inhibited the biological activity of the cells (Li et al. 2015).

Figure 2

Reduction of 30, 70, 140, and 280 mg·L−1 of NO3-N in setups containing ferrihydrite (a), magnetite (b), S. oneidensis MR-1 (c), S. oneidensis MR-1 incubated with magnetite (d), and S. oneidensis MR-1 incubated with ferrihydrite (e). C/C0 represents the ratio of the reduced NO3-N to the initial concentration of NO3-N added.

Figure 2

Reduction of 30, 70, 140, and 280 mg·L−1 of NO3-N in setups containing ferrihydrite (a), magnetite (b), S. oneidensis MR-1 (c), S. oneidensis MR-1 incubated with magnetite (d), and S. oneidensis MR-1 incubated with ferrihydrite (e). C/C0 represents the ratio of the reduced NO3-N to the initial concentration of NO3-N added.

Close modal

In the presence of both iron oxide (either ferrihydrite or magnetite) and S. oneidensis MR-1 bacteria, reduction of nitrate with different dosages was detected (Figure 2(d) and 2(e)). In both systems, the removal efficiency of NO3-N was increased to more than 95% within 10 h at initial concentrations of 30 mg L−1 and 70 mg L−1. When NO3-N concentration was 30 mg·L−1, the final removal efficiencies of NO3-N were 99% and 98.20% in the system for S. oneidensis MR-1 bacteria with ferrihydrite and magnetite, respectively. Compared to the removal efficiency of nitrate (92.25%) in a single microbial system (Figure 2(c)), the removal efficiency was indeed improved, suggesting that the addition of iron minerals greatly participated in the nitrate removal process (Zhu et al. 2013).

Formation of biogenic Fe(II) during the Fe(III) bio-reduction process

Biogenic Fe(II) accumulation synchronized with Fe(III) reduction. To evaluate the influence of nitrate on S. oneidensis MR-1 reduced iron minerals, a set of experiments of Fe(III) bio-reduction in the presence or absence of nitrate was performed (Figure 3). Here, 30 mg·L−1 NO3-N was added in the experiments for the purpose of achieving high nitrate removal efficiency with a low initial concentration and being consistent with the previous study for nitrite (Lu et al. 2017). The average rates of biogenic Fe(II) formation were 4.70 ± 0.12 (mg·L−1) h−1 for ferrihydrite and 1.51 ± 0.01 (mg·L−1) h−1 for magnetite within 34 h. In the system containing ferrihydrite and S. oneidensis MR-1 bacteria, biogenic Fe(II) concentration was about three times higher than that from magnetite bio-reduction, indicating that the difference in particle size and crystallinity can influence the extent of Fe(III) bio-reduction (Xie et al. 2017). It was noted that, in the presence of NO3-N, the reduction of both ferrihydrite and magnetite, as mediated by S. oneidensis MR-1 bacteria, hardly occurred in the first 10 h. This was attributable to the preferred reduction of NO3-N by S. oneidensis MR-1 bacteria over Fe(III) (Lovley et al. 2004). However, in both cases the concentration of biogenic Fe(II) gradually increased after 10 h and reached the same maximum and at about the same time as before the addition of nitrate. These results showed that the addition of NO3-N was able to delay the onset of biogenic Fe(II) formation, but did not prevent the system from reaching the same final Fe(II) concentration or in a same length of time. In other words, there was no inhibition effect observed for NO3-N on Fe(III) bio-reduction in this work.

Figure 3

The accumulation of total Fe(II) in setups containing S. oneidensis MR-1 incubated with iron minerals (with or without adding nitrate) with incubation time.

Figure 3

The accumulation of total Fe(II) in setups containing S. oneidensis MR-1 incubated with iron minerals (with or without adding nitrate) with incubation time.

Close modal

Nitrate removal in multiple redox cycles of Fe(III) bio-reduction

In the system for iron oxide (either ferrihydrite or magnetite) associated with S. oneidensis MR-1 bacteria, three periods of nitrate reduction were completed within 170 h (Figure 4). During the first period in the first 30 h, biogenic Fe(II) was generated gradually in both systems accompanied by the reduction of nitrate. The Fe(II) concentration reached its maximum at 45 h at 193.47 ± 4.73 mg·L−1 and 95.93 ± 10.09 mg·L−1 for the ferrihydrite and magnetite systems, respectively. In the second period, the oxidation of Fe(II) to Fe(III) coupled with the reduction of NO3 commenced, followed by a gradual reduction in Fe(III) by S. oneidensis MR-1 bacteria that resulted in a relatively high concentration of Fe(II) before the third period started. By comparison, in the control experiments, the reduction of NO3 by S. oneidensis MR-1 bacteria took more than 110 h during the second period (Fig. S1). In contrast, nitrate was removed three times during the biotic process in 220 days in the coculture system of DIRB/nitrate-dependent Fe(II)-oxidization, which had a long removal cycle (Zhao et al. 2015). However, after the last nitrate addition at the beginning (4 h) of the third period, the rate of nitrate removal was significantly decreased. The removal rates of nitrate in the three stages were 3.61 mg·L−1·h−1, 1.98 mg·L−1·h−1, and 1.01 mg·L−1·h−1 in the ferrihydrite system, respectively, and 4.13 mg·L−1·h−1, 2.36 mg·L−1·h−1, and 1.53 mg·L−1·h−1 in the magnetite system. Furthermore, it was noted that dissimilatory iron reduction by S. oneidensis MR-1 bacteria was not observed after three periods of NO3 removal, similarly shown by previous studies (Zhao et al. 2015). In comparison with the previous work on continuous nitrite removal (Lu et al. 2017), the number of periods for nitrate removal under the same conditions decreased. A possible reason was the accumulation of nitrite as an intermediate product of nitrate reduction. The bio-toxicity of nitrite, combined with cell encrustation caused by the abiotic reaction of Fe(II) and nitrite, may have resulted in cell death or non-growth of the S. oneidensis MR-1 bacteria population (Zhao et al. 2013).

Figure 4

Time-course changes for NO3-N and Fe(II) through multiple redox cycles associated with ferrihydrite (a) and magnetite (b) reduction by S. oneidensis MR-1. The arrows indicate the addition of nitrate (30 mg·L−1).

Figure 4

Time-course changes for NO3-N and Fe(II) through multiple redox cycles associated with ferrihydrite (a) and magnetite (b) reduction by S. oneidensis MR-1. The arrows indicate the addition of nitrate (30 mg·L−1).

Close modal

Contribution of biotic and abiotic reductions for nitrate removal

To investigate the role of biotic and abiotic processes in nitrogen transformation, reductive transformation of NO3-N in both iron oxide/S. oneidensis MR-1 bacteria systems during the first and second nitrate reduction periods was analyzed (Figure 5). In the first period, the NO3-N removal efficiencies were 94.4% for ferrihydrite and 98.1% for magnetite, while 99% of the final product almost was NH4+-N, similar to the results in the control experiment with only the microbe present (Table 1). Notably, NO2-N accumulated as an intermediate at the beginning of the reaction (Figure 5(a) and 5(b)), indicating that the reduction pathway from NO3-N to NH4+-N by S. oneidensis MR-1 bacteria was a stepwise process with NO2-N as an intermediate (Yoon et al. 2015). Upon the second addition of nitrate, biogenic Fe(II) produced from the first addition reacted with both of the newly added NO3-N and NO2-N as an intermediate (Figure 5(c) and 5(d)).

Table 1

Content of various types of nitrogen (% of total N) after nitrate reduction by S. oneidensis MR-1 only and in the ferrihydrite- and magnetite-associated S. oneidensis MR-1 systems at the end of the first (1st) and second (2nd) nitrate addition periods

SystemCt/C0 (%)
NO3-NNH4+-NNO2-NN2ONO2
S. oneidensis MR − 1 3.11 96.12 0.77 NDa ND 
Ferrihydrite/S. oneidensis MR − 1 (1st) 5.64 93.56 0.80 ND ND 
Magnetite/S. oneidensis MR − 1 (1st) 1.93 97.57 0.50 ND ND 
Ferrihydrite/S. oneidensis MR − 1 (2nd) 14.44 29.15 9.08 25.14 22.19 
Magnetite/S. oneidensis MR − 1 (2nd) 10.94 62.15 10.11 10.13 6.67 
SystemCt/C0 (%)
NO3-NNH4+-NNO2-NN2ONO2
S. oneidensis MR − 1 3.11 96.12 0.77 NDa ND 
Ferrihydrite/S. oneidensis MR − 1 (1st) 5.64 93.56 0.80 ND ND 
Magnetite/S. oneidensis MR − 1 (1st) 1.93 97.57 0.50 ND ND 
Ferrihydrite/S. oneidensis MR − 1 (2nd) 14.44 29.15 9.08 25.14 22.19 
Magnetite/S. oneidensis MR − 1 (2nd) 10.94 62.15 10.11 10.13 6.67 

aND means not detected.

Figure 5

Reductive transformation of NO3-N in ‘S. oneidensis MR-1/ferrihydrite’ (a) and ‘S. oneidensis MR-1/magnetite’ (b) systems during the first cycle and both of them underwent the second cycle for (c) and (d), respectively. Ng is the sum of N2O and NO2, TN is the sum of NO3-N, NO2-N, NH4+-N and Ng.

Figure 5

Reductive transformation of NO3-N in ‘S. oneidensis MR-1/ferrihydrite’ (a) and ‘S. oneidensis MR-1/magnetite’ (b) systems during the first cycle and both of them underwent the second cycle for (c) and (d), respectively. Ng is the sum of N2O and NO2, TN is the sum of NO3-N, NO2-N, NH4+-N and Ng.

Close modal

As shown in Table 1, in the ferrihydrite/S. oneidensis MR-1 bacteria system, 85.6% NO3-N was removed and gaseous nitrogen (Ng) emission was 47.3% of the initial total N in the second period. The Ng emission contained 25.1% N2O and 22.2% NO2 according to GC analysis (Fig. S2). In comparison, NO3-N removal efficiency was 89.1% and yield of Ng was 16.8% (contained 10.1% N2O and 6.67% NO2) in the magnetite/S. oneidensis MR-1 bacteria system. Moreover, both systems contained around 10% of NO2-N, which might have brought negative effects on NO3-N removal. Given that total nitrogen in solution in the first period remained the same, indicating no gaseous nitrogen production, it was reasonable to believe abiotic nitrate/nitrite reduction by biogenic Fe(II) contributed to all the gaseous nitrogen production in the second nitrate addition period.

In the first period, both nitrate and Fe(III) can be used as electron acceptor for the growth of DIRB. NH4+-N and biogenic Fe(II) gradually formed as the product of microbial nitrate and Fe(III) reduction, respectively. These two reactions were biotic processes. Microbial-mediated Fe(III) reduction is the key for the start-up of redox cycle in the first period. It can be seen from Fig. S3 that Fe2+ prepared by dissolution of FeSO4·4H2O, did not react abiotically with nitrate. Therefore, biogenic Fe(II) generated from the biotic process played a dominant role in nitrogen removal from the system.

Mineral conversion

Change of ferrihydrite and magnetite during redox cycling of nitrate was measured by XRD (Fig. S4). The XRD pattern after 170 h reaction of ferrihydrite shows the characteristic peak of magnetite. The results demonstrated that ferrihydrite is gradually converted into magnetite during the process of microbial-mediated Fe(III) reduction. Magnetite, on the other hand, remained structurally consistent throughout. Fe(II) obtained by the reduction of structural state Fe(III) by iron-reducing bacteria would undergo a series of complex reactions with iron (hydrogen) oxides, including adsorption, electron transfer, reduction dissolution, atomic exchange and crystal phase recombination, thus transformed to secondary minerals (Zachara et al. 2002). The biogenic Fe(II) was oxidized with the added NO3-N, thus completing one iron redox cycle. Therefore, it was the biotic reduction of Fe(III) that started the iron redox cycle. Reaction equations of these redox cycles were consistent with the previously published research (Lu et al. 2017). As a whole, both iron minerals showed excellent cycling.

Relationship and difference between nitrate and nitrite

S. oneidensis MR-1 can use nitrate and nitrite as its electron acceptor, and the product is NH4+-N (Cruz-Garcia et al. 2007; Simpson et al. 2010). In nitrate removal by biotic Fe(II), however, it is necessary to first convert nitrate into nitrite (Cooper et al. 2003). That will lead to the accumulation of part of the nitrite produced, and a longer removal time for nitrate than nitrite. Using the same experimental system, we previously demonstrated that a reduction in iron could still be observed when the reaction time was 180 h, which indicated that biological reduction was still in progress (Lu et al. 2017). In the current experiment, staining of living and dead cells showed a large number of active microorganisms within 72 h after the end of the experiment (Figure 6). After the third addition of nitrate, no reduction of iron minerals was observed, indicating that the accumulation of nitrite inhibited the activity of S. oneidensis MR-1. A high NH4+-N concentration will also inhibit the activity of microorganisms (Zhang et al. 2020). Therefore, the effective removal of nitrate, like nitrite, depends on the presence of biogenic Fe(II) but, in the cycling system, the accumulation of nitrite and NH4+-N inhibits the activity of S. oneidensis MR-1, thus hindering iron reduction. Although the removal of nitrate was different from that of nitrite, after the iron reduction process, ferrihydrite was transformed into magnetite, and magnetite still maintained its own crystal structure.

Figure 6

Staining images of S. oneidensis MR-1 after the end of the experimental cycle system. The upper part is the ferrihydrite system and the lower part is the magnetite system.

Figure 6

Staining images of S. oneidensis MR-1 after the end of the experimental cycle system. The upper part is the ferrihydrite system and the lower part is the magnetite system.

Close modal

In this study, continuous nitrate removal at a low concentration (30 mg·L−1) using the iron redox cycle mediated by S. oneidensis MR-1 bacteria was investigated. It was found that nitrate was removed continuously. Microbial-mediated Fe(III) reduction was the key for the start-up of the redox cycle. The Fe(II) concentration reached its maxima at 45 h at 193.47 ± 4.73 mg·L−1 and 95.93 ± 10.09 mg·L−1 for the ferrihydrite and magnetite systems, respectively. The removal mechanism of nitrate was the same as that of nitrite, i.e. abiotic oxidization of biogenic Fe(II) coupled with the reduction in NO3/NO2 was the sole process responsible for the removal of TN. The difference was that, in the nitrate system, especially after the third addition of nitrate, reduction of iron minerals can no longer be observed due to the accumulation of the intermediate product nitrite from nitrate reduction. However, biogenic Fe(II) produced from the initial microbial-mediated Fe(III) reduction reacted with nitrite to remove it from the system. Time allowed for this reaction may be increased to complete the removal of nitrite before the addition of more nitrate. Overall, the excellent Fe3+/Fe2+ cycling of the two iron oxides tested in this study provide a potential application for continuous nitrate removal in the aquatic environment.

This work was financially supported by the Program of Intergovernmental Cooperation in Science and Technology (2017YFE0109800), National Natural Science Foundation of China (No. 41877187) and Program for Innovative Research Team in University (No. IRT13078).

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

Cao
S.
Zhou
Y.
2019
New direction in biological nitrogen removal from industrial nitrate wastewater via anammox
.
Applied Microbiology and Biotechnology
103
(
18
),
7459
7466
.
Cooper
D. C.
Picardal
F. W.
Schimmelmann
A.
Coby
A. J.
2003
Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200
.
Applied and Environmental Microbiology
69
(
6
),
3517
3525
.
Cruz-Garcia
C.
Murray
A. E.
Klappenbach
J. A.
Stewart
V.
Tiedje
J. M.
2007
Respiratory nitrate ammonification by Shewanella oneidensis MR-1
.
Journal of Bacteriology
189
(
2
),
656
662
.
Howarth
R. W.
2004
Human acceleration of the nitrogen cycle: drivers, consequences, and steps toward solutions
.
Water Science and Technology
49
(
5–6
),
7
13
.
Hu
Y.-R.
Guo
C.
Wang
F.
Wang
S.-K.
Pan
F.
Liu
C.-Z.
2014
Improvement of microalgae harvesting by magnetic nanocomposites coated with polyethylenimine
.
Chemical Engineering Journal
242
,
341
347
.
Kappler
A.
Bryce
C.
Mansor
M.
Lueder
U.
Byrne
J. M.
Swanner
E. D.
2021
An evolving view on biogeochemical cycling of iron
.
Nature Reviews Microbiology
19
,
360
374
.
Li
B.-B.
Cheng
Y.-Y.
Wu
C.
Li
W.-W.
Yang
Z.-C.
Yu
H.-Q.
2015
Interaction between ferrihydrite and nitrate respirations by Shewanella oneidensis MR-1
.
Process Biochemistry
50
(
11
),
1942
1946
.
Lovley
D. R.
Phillips
E. J.
1986
Availability of ferric iron for microbial reduction in bottom sediments of the freshwater tidal potomac river
.
Applied and Environmental Microbiology
52
(
4
),
751
757
.
Lovley
D. R.
Holmes
D. E.
Nevin
K. P.
2004
Dissimilatory Fe(III) and Mn(IV) reduction
.
Advances in Microbial Physiology
49
,
219
286
.
Lu
Y.
Xu
L.
Shu
W.
Zhou
J.
Chen
X.
Xu
Y.
Qian
G.
2017
Microbial mediated iron redox cycling in Fe (hydr)oxides for nitrite removal
.
Bioresource Technology
224
,
34
40
.
Lu
Y.
Huang
X.
Xu
L.
Chen
X.
Zhou
J.
Qian
G.
Fang
F.
2020
Elucidation of the nitrogen-transformation mechanism for nitrite removal using a microbial-mediated iron redox cycling system
.
Journal of Water Process Engineering
33
, 101016.
Marsili
E.
Baron
D. B.
Shikhare
I. D.
Coursolle
D.
Gralnick
J. A.
Bond
D. R.
2008
Shewanella secretes flavins that mediate extracellular electron transfer
.
Proceedings of the National Academy of Sciences of the United States of America
105
(
10
),
3968
3973
.
Melton
E. D.
Swanner
E. D.
Behrens
S.
Schmidt
C.
Kappler
A.
2014
The interplay of microbially mediated and abiotic reactions in the biogeochemical Fe cycle
.
Nature Reviews Microbiology
12
(
12
),
797
808
.
Pan
Y.
Yang
X.
Sun
G.
Xu
M.
2019
Functional response of sediment bacterial community to iron-reducing bioaugmentation with Shewanella decolorationis S12
.
Applied Microbiology and Biotechnology
103
(
12
),
4997
5005
.
Park
H.
Park
Y.-M.
Yoo
K.-M.
Lee
S.-H.
2009
Reduction of nitrate by resin-supported nanoscale zero-valent iron
.
Water Science and Technology
59
(
11
),
2153
2157
.
Peretyazhko
T. S.
Zachara
J. M.
Kennedy
D. W.
Fredrickson
J. K.
Arey
B. W.
McKinley
J. P.
Wang
C. M.
Dohnalkova
A. C.
Xia
Y.
2010
Ferrous phosphate surface precipitates resulting from the reduction of intragrain 6-line ferrihydrite by Shewanella oneidensis MR-1
.
Geochimica et Cosmochimica Acta
74
(
13
),
3751
3767
.
Roden
E. E.
Urrutia
M. M.
2002
Influence of biogenic Fe(II) on bacterial crystalline Fe(III) oxide reduction
.
Geomicrobiology Journal
19
(
2
),
209
251
.
Suzuki
T.
Moribe
M.
Oyama
Y.
Niinae
M.
2012
Mechanism of nitrate reduction by zero-valent iron: equilibrium and kinetics studies
.
Chemical Engineering Journal
183
,
271
277
.
Wang
F.
Zheng
S.
Qiu
H.
Cao
C.
Tang
X.
Hao
L.
Liu
F.
Li
J.
2018
Ferrihydrite reduction and vivianite biomineralization mediated by iron reducing bacterium Shewanella oneidensis MR-4
.
Acta Microbiologica Sinica
58
(
4
),
573
583
.
Wu
J.
2020
Challenges for safe and healthy drinking water in China
.
Current Environmental Health Reports
7
,
292
302
.
Xie
Y.
Dong
H.
Zeng
G.
Tang
L.
Jiang
Z.
Zhang
C.
Deng
J.
Zhang
L.
Zhang
Y.
2017
The interactions between nanoscale zero-valent iron and microbes in the subsurface environment: a review
.
Journal of Hazardous Materials
321
,
390
407
.
Zachara
J. M.
Kukkadapu
R. K.
Fredrickson
J. K.
Gorby
Y. A.
Smith
S. C.
2002
Biomineralization of poorly crystalline Fe(III) oxides by dissimilatory metal reducing bacteria (DMRB)
.
Geomicrobiology Journal
19
(
2
),
179
207
.
Zhang
B.
Deng
J.
Chen
Y.
Meng
D.
Tan
Z.
2020
Effect and quantitative characterization of free ammonia on nitrification process
.
Chinese Journal of Applied and Environmental Biology
26
(
5
),
1260
1267
.
Zhao
L.
Dong
H.
Kukkadapu
R.
Agrawal
A.
Liu
D.
Zhang
J.
Edelmann
R. E.
2013
Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. strain 2002
.
Geochimica et Cosmochimica Acta
119
,
231
247
.
Zhao
L.
Dong
H.
Kukkadapu
R. K.
Zeng
Q.
Edelmann
R. E.
Pentrak
M.
Agrawal
A.
2015
Biological redox cycling of iron in nontronite and Its potential application in nitrate removal
.
Environmental Science & Technology
49
(
9
),
5493
5501
.
Zhou
G.-W.
Yang
X.-R.
Li
H.
Marshall
C. W.
Zheng
B.-X.
Yan
Y.
Su
J.-Q.
Zhu
Y.-G.
2016
Electron shuttles enhance anaerobic ammonium oxidation coupled to iron(III) reduction
.
Environmental Science & Technology
50
(
17
),
9298
9307
.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY-NC-ND 4.0), which permits copying and redistribution for non-commercial purposes with no derivatives, provided the original work is properly cited (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Supplementary data