Tuo
Wang
a,
Jiayao
Zhang
a,
Ziyuan
Wang
a,
Qian
Zhao
a,
Yue
Wu
a,
Nan
Li
b,
Xinlei
Jiang
*c and
Xin
Wang
*a
aMOE Key Laboratory of Pollution Processes and Environmental Criteria/Tianjin Engineering Center of Environmental Diagnosis and Contamination Remediation, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China. E-mail: xinwang1@nankai.edu.cn; Fax: +(86)22 23501117; Tel: +(86)18722292585
bSchool of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China
cSchool of Environmental and Municipal Engineering, Tianjin Chengjian University, Tianjin 300384, P. R. China. E-mail: jiangxinlei5586@126.com
First published on 19th April 2024
Autotrophic anaerobic ammonium oxidation coupled to Fe(III) reduction (Feammox) is a potential technology for removing ammonium from low-C/N wastewater, but it requires a continuous supply of Fe(III) source. To reduce the supply, a microbial electrolysis cell (MEC) was employed to allow iron recycling in Feammox under different voltages (0.2 V, 0.6 V, and 1.0 V). Results showed that the optimal voltage was 0.6 V, with a maximum efficiency for ammonium oxidation of 71%. The ammonium oxidation rate achieved 2.5 ± 0.1 mg N L−1 per day, which was 3 times that of conventional Feammox. Cyclic voltammetry confirmed that ammonium oxidation and iron redox occurred on the anode. The bacterial population had a unique evolutionary direction at 0.6 V, with Geobacteraceae becoming the dominant family. Positive interactions between nitrogen-related bacteria and iron-related bacteria enhanced the autotrophic Feammox process. This study will further advance Feammox in the treatment of ammonium-containing wastewater.
Water impactAutotrophic Feammox has attracted a lot of interest in the treatment of low-C/N wastewater. However, incessant supplementation with Fe(III) sources is required for Feammox in engineering. This study adopted an MEC to drive the iron cycle and promote the ammonium oxidation rate of Feammox. The results revealed the potential mechanism of the iron cycle and bacterial interactions, providing valuable research ideas and technological references for Feammox in engineering. |
NH4+ + 3Fe(OH)3 + 5H+ → 3Fe2+ + 9H2O + 0.5N2 ΔrGm = −245 kJ mol−1 | (1) |
NH4+ + 6Fe(OH)3 + 10H+ → 6Fe2+ + 16H2O + NO2− ΔrGm = −164 kJ mol−1 | (2) |
NH4+ + 8Fe(OH)3 + 14H+ → 8Fe2+ + 21H2O + NO3− ΔrGm = −207 kJ mol−1 | (3) |
The rate of ammonium oxidation has a strong positive correlation with Fe(III) reduction in the Feammox process (Pearson correlation coefficient 0.97, P < 0.01).9 This means that good ammonium removal by Feammox requires a large amount of Fe(III) sources. The Fe(III) resources of Feammox are diverse, including FeCl3, Fe2O3, Fe3O4, Fe(OH)3, ferrihydrite, and goethite.10,11 However, they are always limited in wastewater treatment systems. A sufficient supplement of Fe(III) is necessary to sustain Feammox, which undoubtedly increases the cost of wastewater treatment, making it hard to utilize directly at a large scale. The recycling of iron is one of the most applicable ways to solve this problem, with the interconversion of Fe(III) and Fe(II). Fe(III) can be reduced to Fe(II) by the iron reduction bacteria of Feammox; thus, the point of the iron cycle is the oxidation of Fe(II) to Fe(III). Common methods for oxidizing Fe(II) involve supplying O2 and coupling to a bioprocess. Within the tolerance range of Feammox to oxygen, constant micro-aeration or intermittent aeration were adopted to provide a low-oxygen environment, which achieved Fe(II) oxidation and further enhanced Feammox performance.12,13 In addition, the most frequently mentioned bioprocess is nitrate-dependent Fe(II) oxidation (NDFO). Fe(II) is oxidized by nitrate, coupled with Feammox to implement the iron cycle by supplying nitrate.14 However, these methods demand rigorous control over oxygen and incessant input of nitrate, which makes Feammox complicated and uneconomic in practice. Therefore, other alternatives to the iron cycle are worth exploring.
By inserting a pair of polarized inert electrodes, the redox environment for electroactive microbes can be regulated by an external power source, while the system is known as a microbial electrolysis cell (MEC).15 MEC is an ideal technology for superseding O2 and nitrate due to its low cost, long-term sustainability, simple operation and environmental friendliness. Although Feammox in the MEC has been studied, the role of the iron cycle in the system has rarely been studied. For example, the ammonium oxidation rate of Feammox was improved with continuous supplementation by Fe2O3, which might be the effect of the iron cycle in the MEC system.16 Therefore, exploring the role of the MEC in the iron cycle is essential for understanding the mechanism of the improvement, which has not yet been investigated. MEC may stimulate microbes involved in the iron cycle or directly participate in the redox reaction of iron on the electrodes.
Here, electrochemically assisted Feammox (shortened to E-Feammox) was constructed, and the feasibility of in situ electron supplementation to the Feammox microbial community was demonstrated. The role of the iron cycle and functional bacteria under the different voltages were investigated, and the mechanism was studied according to microbial electrochemical tests, biofilm morphology and microbial community analysis.
The voltages were set at 0.2 V, 0.6 V and 1.0 V, respectively, to MECs, which were lower than the oxygen evolution potential of carbon felt according to a linear sweep voltammetry (LSV) test (Fig. S2†). The experimental controls included abiotic MECs (ck-0.2, ck-0.6 and ck-1.0, corresponding to the different voltages) and a biological control without voltage (ck-bio). All MECs were performed in duplicate at room temperature, avoiding light. When sampling, N2 bags were used for replacement to prevent O2 permeation and to maintain the pressure balance.
To determine E-Feammox reactions on the anode, cyclic voltammetry (CV) was conducted using a potentiostat (Autolab PGSTAT 302 N, Metrohm, Switzerland) in a three-electrode system. Ag/AgCl (0.197 V vs. SHE) with saturated KCl was used as the reference electrode. The CV curve was scanned from −1.0 V to 1.0 V at a rate of 1.0 mV s−1.22 The procedure for measuring oxidation peak currents (Ipa) involved drawing a tangent line along the curve to the peak bottom, and a perpendicular line from the peak top to tangent line. The height of this line represented the Ipa value. Charge and current were used to assess the electron transfer to the anode during Feammox, which were calculated as in eqn (4),23
C = ΔNH4+·n·v·F | (4) |
Biofilms attached to the anode were collected at the end of the experiment to analyze the bacterial community under the different voltages. Bacterial analysis used an Illumina MiSeq sequencing platform by Biomarker Technologies (Beijing, China). The V3–V4 region of the 16S rRNA gene was amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The operational taxonomic units (OTUs) were identified at 97% similarity level. OTU sequences were annotated using a conservative OTU filtration 0.0050% threshold. Clean read assembly was performed using the DADA2 package. The within-habitat diversity and between-habitat diversity of the bacterial community were examined through alpha and beta diversity analysis. The correlation network between genera was constructed by Spearman rank correlation analysis (p-value < 0.050) and exhibited with Gephi 0.10.1. Functional classification statistics of COG (clusters of orthologous groups of proteins) were conducted using the reconstruction of unobserved states (PICRUSt2) to predict the potential functions of E-Feammox bacteria.
The oxidation of ammonium gradually accelerated after 15 days. The ammonium oxidation rates at 0.2 V, 0.6 V and 1.0 V were 2.0 ± 0.2 mg N L−1 per day, 2.5 ± 0.1 mg N L−1 per day and 2.1 ± 0.2 mg N L−1 per day, which were 2.4–3.0 times that of conventional Feammox [no voltage, 0.82 mg N L−1 per day].18 While the concentration of ammonium was constant in ck-bio, indicating that the ammonium oxidation by E-Feammox was promoted in the MECs. The highest ammonium oxidation rate was achieved at 0.6 V, followed by 1.0 V and 0.2 V, with anodic potentials (vs. SHE) of 0.64 ± 0.01 V, 0.77 ± 0.1 V and 0.22 ± 0.2 V. These potentials were also within the range (−0.2 to 0.8 V vs. SHE) reported in other literature for anodic ammonium oxidation.24–26 As reported in previous studies, the bacteria growing on the anode were impacted by the electrode potential.27 Low potential was not sufficient to support the metabolism of the functional bacteria, while a high potential could destroy the structure of the cell membrane.28 Therefore, in this study, 0.6 V was a suitable voltage for the growth of E-Feammox bacteria. On the 36th day, ammonium concentrations under different voltages were 2 ± 0.2 mM, 2 ± 0.1 mM and 2 ± 0.4 mM, with corresponding removal efficiencies of 59 ± 2%, 71 ± 4% and 61 ± 6%. In contrast to biotic MECs, abiotic ck-0.2, ck-0.6 and ck-1.0 showed no significant ammonium oxidation over 36 days, further confirming that the ammonium was removed biologically.
Conventional Feammox oxidizes ammonium into three metabolic products, that is N2, nitrite and nitrate [see eqn (1)–(3)], and diverse processes yielded different products. For example, A6 bacteria oxidized ammonium to nitrite instead of N2 or nitrate,29 while Feammox products were nitrite and nitrate with activated sludge as the inoculant.30 For the microbial community utilized in this study, the major product of Feammox was N2.18 However, the end products of E-Feammox turned into nitrite and a little nitrate in the MEC system. The production ratios of nitrite were 57 ± 10%, 82 ± 2% and 65 ± 1%, and the ratios of nitrate were 2 ± 0.3%, 4 ± 1.6% and 2 ± 2.4% under 0.2 V, 0.6 V and 1.0 V, respectively. The possible intermediate (NH2OH) was not detected throughout the experiment, so that the main nitrogen loss should be attributed to microbial assimilation and N2 production.18 A similar trend in nitrogen conversion was observed from days 36 to 66. The rate of ammonium oxidation at 0.6 V was 2.2 mg N L−1 per day and was higher than that at 1.0 V [1.9 mg N L−1 per day] or 0.2 V [1.2 mg N L−1 per day]. The accumulation of nitrite still occurred after ammonium oxidation. On the 66th day, the levels of nitrite under above voltages were 3.4 ± 0.3 mM, 2.0 ± 0.5 mM and 1.6 ± 0.2 mM. On the 5th day of the second period, there was a significant ammonium oxidation phenomenon, indicating that the E-Feammox system can be initiated rapidly in the new period. In brief, the polarized anode altered the end products of E-Feammox to mainly nitrite, providing a possibility of combination of Feammox with anammox for autotrophic nitrogen removal.31
Fig. 2 Iron contents (A), X-ray diffraction spectra (B) and X-ray photoelectron spectroscopy diagram (C) of iron precipitates at the start and end of the experiment. |
XRD and XPS analyses of the iron precipitates were carried out to explore the conversion of Fe(II) and Fe(III). There was a high content of vivianite in the inoculant at the beginning of the experiment (Fig. 2B). A reasonable explanation was that the iron reduction bacteria in the microbial community, such as Geobacter, reduced Fe(III) to Fe(II). Then, Fe(II) was chemically precipitated with phosphate as vivianite, which is an important form of P-containing mineral in anaerobic sludge.32,33 The redox potential of vivianite was close to 0 V as per the Eh-pH diagram.34 In the present study, the anodic potentials were all greater than 0 V, which could facilely oxidize vivianite. Thus, the process of Fe(II) oxidation in the iron cycle was achieved by the MEC. The predominant mineral in ck-bio was leucophosphite with Fe(III).35 The contents of vivianite and leucophosphite in the precipitates were different under the applied voltages, confirming the presence of the iron cycle in the MECs. The chemical state of iron in mineral crystals was further investigated through XPS analysis (Fig. 2C). The mineral crystals had characteristic peaks near 711 eV and 725 eV, corresponding to Fe2p3/2 and Fe2p1/2, respectively.36 Based on semi-quantitative fitting of iron, divalent iron accounted for 44% of the total iron in the crystals at 0.6 V, which was higher than that at 0.2 V (33%), 1.0 V (30%) or ck-bio (28%). These results supported the conversion of Fe(II) and Fe(III) in the form of vivianite and leucophosphite on the surface of the anode. The cycling of iron was successfully driven by the MEC system, which significantly enhanced the performance of E-Feammox.
Fig. 4 Confocal laser scanning microscopy images (A), active and inactive cells ratio (B), and cluster tree and relative abundance at the family level (C) of the different biofilms. |
The colonies on the anodes under 0.2 V, 0.6 V, 1.0 V and ck-bio (Fig. 4C) were different, indicating that the applied voltages influenced the evolution of E-Feammox bacteria. The evolutionary direction of the Feammox bacteria under 0.6 V was significantly different from that at 0.2 V or1.0 V. The diverse performance of E-Feammox can be attributed to the different bacterial communities. The bacterial community in the different MECs was analyzed at the family level. The relative abundances of Geobacteraceae and Desulfobulbaceae, which are typical iron reduction bacteria, at 0.6 V were 23% and 13%.39 However, Geobacteraceae and Desulfobulbaceae were not detected with no voltage control, suggesting that MEC promoted the multiplication of the iron reduction bacteria. The high abundance of the iron reduction bacteria further resulted in the highest content of Fe(II) in iron precipitates at 0.6 V. Xanthomonadaceae (1.9%), Comamonadaceae (1.9%) and Hydrogenophilaceae (0.64%) are known bacteria with ammonium oxidation ability.40 These bacteria dominated ammonium oxidation in the E-Feammox process. Brucellaceae (0.50%), Phyllobacteriaceae (0.86%) and Hyphomicrobiaceae (1.3%) are also associated with the nitrogen cycle,41 such as denitrification, which is a potential pathway for producing N2 due to the presence of nitrite. The high E-Feammox rate at 0.6 V should be attributed to the co-metabolism of a high abundance of the iron reduction bacteria and various nitrogen-related bacteria, in which Geobacteraceae play a crucial role. The families at 0.2 V and 1.0 V exhibited a relatively high abundance of Xanthomonadaceae, Comamonadaceae and Hydrogenophilaceae, explaining their capacity to oxidize ammonium. The presence of Geobacteraceae (10%) at 0.2 V was higher than at 1.0 V (0.92%), indicating that high potential was unfavorable for the growth of Geobacteraceae. The abundance of unclassified_Gemmatimonadetes at 1.0 V (7.6%) and no voltage (17%) was high, and they are frequently found in soil and potentially associated with Feammox.42
The metabolic interactions between E-Feammox bacteria were further analyzed at the genus level. There were three main interactions among genera (Fig. 5A). First, Geobacter with the highest weight was positively correlated with unclassified_Desulfobulbaceae and unclassified_Thiobacterales, associated with Fe(III) reduction and extracellular electron transfer in the MEC. The positive correlation between unclassified_Gemmatimonadetes, Bacillus, Chryseobacterium and Dokdonella suggested interaction among them in the nitrogen cycle.43,44 However, they exhibited a negative correlation with Geobacter. The negative interaction may be attributed to niche competition. Second, the ammonium oxidation genus Nitrosomonas formed a positive interaction with denitrification genera Thermomonas, Alicycliphilus and Pseudomonas,45 indicating that the denitrification process was actuated when ammonium was oxidized to nitrite. The third interaction was also related to nitrogen metabolism. Ochrobactrum, Clostridium and Achromobacter were all found to have capability for ammonium oxidation,46 but they exhibited a negative correlation with other nitrogen-related genera GOUTA19 and unclassified_Melioribacteraceae, which may be attributed to competition between them for nitrogen sources. Some denitrification bacteria, such as Thermomonas, Pseudomonas47 and Thiobacillus,48 were capable of oxidizing Fe(II) while reducing nitrate or nitrite, and they existed in various interaction networks. The positive interaction among iron reduction bacteria, iron oxidation bacteria, ammonium oxidation bacteria and denitrification bacteria fulfilled their critical roles in the E-Feammox process.
Based on the COG database, more functional proteins under 0.6 V compared to no voltage were analyzed to reveal the impact of the MEC on Feammox bacteria. The proportion of energy production and conversion at 0.6 V (7.6%) was higher than that for ck-bio of 6.4% (Fig. 5B), suggesting that the metabolic activity of the bacteria was more vigorous in the E-Feammox process. The proteins related to cell multiplication have a higher proportion at 0.6 V, such as cell cycle control, cell division, chromosome partitioning (1.2%), translation, ribosomal structure and biogenesis (7.5%), replication, recombination and repair (4.7%). The high multiplication capacity of the bacteria can form thicker biofilms, which also mutually corroborated the results of CLSM. The protein involved in signal transduction mechanisms should be key to the co-metabolism in the bacterial community. The proportion of 0.6 V (4.9%) was higher than that of ck-bio (4.0%), resulting in better interactions between E-Feammox bacteria under 0.6 V. The functional proteins that may be associated with electron transfer were translated more under 0.6 V, including coenzyme transport and metabolism (5.3%), cell motility (2.8%) and intracellular trafficking, and secretion and vesicular transport (3.0%). However, the pathway and mechanism of electron transfer by E-Feammox bacteria need to be further explored.
This study verified the adoption of an MEC to enhance the autotrophic Feammox performance via the iron cycle. Based on the above content, the mechanism of E-Feammox in MEC was proposed, as shown in Fig. 6. Inoculants containing Feammox bacteria and vivianite [Fe(II)] were added to the MEC system. Owing to the anodic potential being higher than the oxidation potential of vivianite and presence of iron oxidation bacteria, vivianite was gradually oxidized to leucophosphite [Fe(III)]. After the accumulation of Fe(III), the E-Feammox process was initiated to oxidize ammonium and reduce Fe(III). The regenerated Fe(II) was then oxidized again to Fe(III) by the anode and iron oxidation bacteria; thus, the cycling of iron was successfully driven in E-Feammox. Due to the positive interaction between iron-related and nitrogen-related bacteria, the iron cycle promoted the rate of the ammonium oxidation process. When ammonium was oxidized to nitrite or nitrate, some denitrification bacteria were awakened to oxidize Fe(II), which is similar to NDFO and is frequently found in the Feammox system.49 Although the NDFO process was weak in the interaction network, it also strengthened the iron cycle. The MEC system enhanced Feammox mainly through following aspects: (1) boosting the metabolism of Feammox bacteria, including growth activity, bacterial interaction and electron transfer, to increase biomass and thick biofilm attachment to the anode; (2) promoting the evolvement of E-Feammox functional bacteria, such as Geobacteraceae, Xanthomonadaceae, Comamonadaceae, Desulfobulbaceae and Brucellaceae, to strengthen ammonium oxidation and the iron cycle; and (3) accelerating the oxidation of Fe(II) to Fe(III) to drive the cycling of iron. Therefore, MEC is a feasible technology for intensifying autotrophic Feammox in the treatment of ammonium-containing wastewater.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ew00074a |
This journal is © The Royal Society of Chemistry 2024 |