Transformation of Nitrogen and Iron Species during Nitrogen Removal

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Transformation of nitrogen and iron species during nitrogen removal from wastewater via Feammox by adding ferrihydrite Yafei Yang, Zhen Jin, Xie Quan, and Yaobin Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03083 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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ACS Sustainable Chemistry & Engineering

Author list Authors: Yafei Yang, Zhen Jin, Xie Quan, Yaobin Zhang*

Dr. Yafei Yang (First author) E-mail address: [email protected] Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province.

Dr. Zhen Jin E-mail address: [email protected] Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province.

Prof. Xie Quan E-mail address: [email protected] Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. 1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province.

Prof. Yaobin Zhang (Corresponding author) E-mail address: [email protected] Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province.

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Transformation of nitrogen and iron species during nitrogen removal from wastewater via Feammox by adding ferrihydrite Yafei Yang, Zhen Jin, Xie Quan, Yaobin Zhang*

Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China

*Corresponding author: Yaobin Zhang; School of Environmental Science and Technology, Dalian University of Technology, Dalian, China; Phone: +86-411-84706140. Fax: +86-411-84706263. E-mail: [email protected].

Abstract Fe(III) reduction coupled to anaerobic ammonium oxidation, namely Feammox, plays an important role in the Fe/N cycle in natural environments. However, it has been rarely studied in wastewater treatment systems. To date, transformation of nitrogen and iron species of Feammox during anaerobic digestion remain unknown. In this study, ferrihydrite was supplemented (50 mM) in an anaerobic digester to remove ammonium through the Feammox. Results showed that the ammonium removal efficiency after 63 days reached 69.49%, significantly higher than that of the control (35.63%). X-ray diffraction analysis showed that the ferrihydrite was transformed into magnetite and akaganeite after the experiment. Further study found that Fe(II) could be oxidized by NOx- in the inoculants taken from the ferrihydrite-supplemented group; i.e., possible NOx--dependent Fe(II) oxidation could provide the possibility for further 3



Feammox. Microbial analysis showed that iron-reducing bacteria and iron-oxidizing bacteria were both detected in two groups. Keywords: Feammox; NOx--dependent Fe(II) oxidation; Anaerobic digestion; Nitrogen loss.

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INTRODUCTION In general, nitrification-denitrification and anaerobic ammonium oxidation (anammox) have been considered as the two main approaches to biological nitrogen removal.1-3 The nitrification-denitrification process operated in separate aerobic and anaerobic tanks is energy-intensive and requires amendment with extra carbon sources in many low C/N wastewaters. The anammox process, which removes ammonium with nitrite as the electron acceptor under anoxic conditions (NH4+ + NO2- → N2 + 2H2O), is sensitive to the environmental conditions, and the proliferation of anammox bacteria is slow.4-6 Recently, Feammox, i.e., ferric iron [Fe(III)] reduction coupled to anaerobic ammonium oxidation, has been reported to play an important role in the nitrogen cycles of natural environments.7-10 In Feammox, Fe(III) is reduced to Fe(II), accompanied with oxidation of NH4+ to N2, NO2- and NO3- (eq. 1-3). Feammox has been estimated to metabolize 7.8−61 (3.9−31% of nitrogen fertilizer loss) kg of NH4+/ha/year in paddy soil.11 In our previous study, it was found that 20.1% total nitrogen removal was achieved when supplementing Fe(OH)3 in a high-ammonium anaerobic sludge digester.12 Although the efficiency was not high, Feammox had showed its potential to run as an in-site anaerobic ammonium treatment process. However, little is known about the transformation and possible interactions of nitrogen and iron species during Feammox as well as the potential effects on the nitrogen removal in anaerobic wastewater treatment. For example, NOx- as a product of Feammox was reported to be capable of oxidizing Fe(II) to Fe(III), termed as

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NOx--dependent Fe(II) oxidation (NDFO).13-15 In other words, Fe(III) is likely regenerated by NDFO. Therefore, the aim of this study was to investigate the effects of Feammox on nitrogen removal, including the following contents: 1) changes in the nitrogen and iron species during Feammox; 2) possible interaction during ferrihydrite related Feammox process and their potential effects on the nitrogen removal; and 3) bacterial communities analysis. 3Fe(OH)3 + 5H+ + NH4+ → 3Fe2+ + 9H2O + 0.5N2 △rGm = -245 KJ/mol

(1)

6Fe(OH)3 + 10H+ + NH4+ → 6Fe2+ + 16H2O + NO2-△rGm = -164 KJ/mol

(2)

8Fe(OH)3 + 14H+ + NH4+ → 8Fe2+ + 21H2O + NO3-△rGm = -207 KJ/mol

(3)

MATERIALS AND METHODS Experimental procedures. Inoculant sludge with a solid content of 10% was taken from a sludge treatment plant in Dalian (China) and stored in a refrigerator at 4 °C. The ferrihydrite used in this study was synthesized by adding Fe(III) chloride to water that was maintained at a pH of 6.8-7.2.16 Morphology of the synthesized ferrihydrite characterized by a scanning electron microscope is shown in Fig.S1. Prior to incubation, the sterile anoxic deionized water (autoclaved at 120 °C for 20 min) was added into the inoculant sludge at a ratio of 5:1 (v/v), and preincubated anoxically in the dark at 25 °C for 1 days statically, then pouring out the supernatant after centrifugation to remove indigenous NH4+ and NOx-. The above procedure was repeated for three times. Then, the prepared sludge after removing supernatant was

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used as the inoculant sludge. Two batch experiments were conducted in 200 mL serum vials: a control group and a ferrihydrite group. 10 mL prepared inoculant sludge was added into each vial as the seed sludge. Ferrihydrite was supplemented in the ferrihydrite group with a dose of 50 mM, while Fe(III) was not supplemented in the control. Then, NH4+-containing medium was added to ensure that the initial volume of each serum vial was 150 mL. The medium (pH 5.0) contains MgCl2·6H2O (0.4 g/L), CaCl2·H2O (0.1 g/L), NH4Cl (0.027 g/L), KH2PO4 (0.6 g/L), 1 ml/L of a vitamin solution, 1 ml/L of a trace element solution and 30 mM bicarbonate buffer.16 Afterwards, the serum vials were sealed with butyl rubber septa, crimped with aluminum caps. The headspace of the vial was flushed with N2 and CO2 (80/20%) for 30 minutes. Then, the vials were incubated statically at 25 °C in the dark for 87 days. The experiments were repeated in triplicate. The initial parameters of the mixture in the control and ferrihydrite groups (before adding ferrihydrite) are shown in Table 1. Anaerobic NDFO experiment was conducted using a homogenized slurry collected from the ferrihydrite group after the above-mentioned experiment. This experiment was operated under the following three culture conditions, respectively: (i) 1 mL slurry+1.2 mM NO3-, (ii) 1 mL slurry+1.2 mM NO2- and (iii) 1 mL slurry+ sterilized deionized water. These three cultures were respectively added in three 100 mL-serum vials containing 50 mL sterilized medium. After flushing the headspace of the vials with N2 and CO2 (80/20%), the vials were incubated statically at 25 °C in the dark for 30 days. Prior to sampling, the vials were thoroughly mixed to ensure the sampling 7



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equally. The experiments were repeated in triplicate. Analysis

Methods.

The

pH

was

determined

with

a

dual-channel

pH-ion-conductivity-dissolved oxygen meter (X60, Fisher Scientific). The total chemical oxygen demand (TCOD) was analyzed according to a reference.17 The contents of protein, polysaccharide and total nitrogen were determined based on the method described in our previous report.12 The well-mixed slurry was sampled inside the anaerobic chamber, and centrifuging (10000 rpm) for 5 min. The supernatant was filtered through a 0.22 µm PTFE filter. The filtrate was then applied for measuring NH4+, NO2- and NO3- using a Dionex DX1000 ion chromatograph (IC) system. The well-mixed slurry was dried in a desiccator located inside the anoxic chamber to avoid oxidation. The dried solids were analyzed by X-ray diffraction (XRD)18, 19 and X-ray photoelectron spectroscopy (XPS)20-22 to identify the crystalline iron phases present in the sample. XRD measurement was carried out via an XRD diffractometer (Empyrean, Panalytical, Netherlands) with Cu-Kα radiation at 45 kV and 200 mA, and XRD pattern was analyzed by the software Jade. XPS measurement was performed using a 250Xi system (EscalabTM 205Xi, Thermofisher, America), and the XPSPEAK4.1 software was used for pattern analysis. Fe(II) and Fe(III) contents in the slurry were analyzed according to the method described by Ding et al.11 In brief, 1 mL of the slurry was immediately extracted with 5 mL of 0.5 M HCl for 2 h at room temperature, and then the mixture was centrifuged at 10000 rpm for 10 min. The Fe(II) and total iron concentrations in the supernatant were measured according to the standard method.23 The amount of Fe(III) was 8


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calculated as the difference between the total iron and Fe(II). All extractions were conducted in an anoxic glovebox. The details of Fe analysis are provided in the Supplemental Information. After the 87-day Feammox experiment, the microbial communities in the two groups were analyzed via high-throughput 16S rRNA pyrosequencing. The detailed methods of DNA extraction, PCR and sequencing are also provided in the Supporting Information.

RESULTS AND DISCUSSION Changes of the nitrogen species during the experiment. Fig.1a demonstrates the changes in the NH4+-N content during the experiment. In the initial 20 days, the NH4+-N content increased drastically from 10.14 to 66.15 mg/L in the ferrihydrite group, while that in the control group increased slightly to 21.82 mg/L, indicating that ferrihydrite accelerated the release of ammonium from the decomposition of proteins that was a major organic component of sludge.24 As shown in Fig.1b, the protein of the ferrihydrite group on day 37 was undetectable, while 21.1 mg/L protein was still observed in the control, meaning that protein removal efficiency was higher with amending ferrihydrite. These results agreed well with previous reports that Fe(III) enhanced the anaerobic decomposition of protein.25 After the first 20 days, the NH4+-N content of the ferrihydrite group exhibited an obvious downward trend to 20.05 mg/L on day 87. In contrast, the NH4+-N content of the control increased to 35.62 mg/L on day 87; this content was higher than that of the ferrihydrite group. 9



Ammonium is an intermediate product of protein degradation that might be consumed by the potential Feammox process, which could explain the fluctuation in the ammonium content observed in the two groups. In addition to ammonium, organic matters could also provide electrons for the microbial dissimilatory Fe(III) reduction. From the thermodynamic point of view, organic substrates are more suitable to use as electron donors in the Fe(III) reduction than ammonium.26 Therefore, as the organic matters were consumed over time, the dissimilatory Fe(III) reduction was more inclined to use ammonium as the electron donor, which could be a main reason for the lag in ammonium removal (Fig.1a). The control group contained 49.85 mg/L of endogenous Fe(III) (Table 1), which also resulted in the nitrogen loss through Feammox, although the nitrogen loss was obviously lower than that in the ferrihydrite group. Notably, the ammonium content of the ferrihydrite group showed less changes during the period from day 36-47, while the content decreased again after sodium bicarbonate (NaHCO3) was added into each serum vial to final content of 1.5 mM on day 49. This change was consistent with Feammox being inorganically autotrophic27, 28

and requiring a sufficient amount of inorganic carbon as the carbon source. To

further verify that ammonium was removed in the presence of ferrihydrite, on the 87th day, an extra 73.2 mg/L of NH4+-N was added to the ferrihydrite group. Meanwhile, an additional bicarbonate was also added into the system as the carbon source (final content was 1.5 mM). At this time, the content of Fe(III) in the ferrihydrite group was 1294.5 mg/L. As shown in Fig.1a, the NH4+-N content dropped again to 54.5 mg/L 10


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after the following 30 days. The pH of the two systems ranged from 5.0-6.2 during the experiment, and these weakly acidic conditions prevented volatilization of the ammonium. An abiotic experiment with no inoculant sludge was conducted in parallel and showed that the ammonium content almost remained unchanging, i.e., 7.43 ± 0.08 mg/L, when adding ferrihydrite alone, further indicating that ammonium removal here was a biotic process (Fig.1c). In Feammox, NH4+ was directly converted to N2 or oxidized to NOx-, which might be further denitrified into N2. Considering that NH4+ is an intermediate in the anaerobic decomposition of protein, the total nitrogen, including organic and inorganic nitrogen of well-mixed slurry, was determined to further clarify the Feammox process. On day 37, the total nitrogen in the control decreased from the initial level of 106.48 to 96.54 mg/L, while the total nitrogen in the ferrihydrite group decreased from 106.48 to 68.01 mg/L (Fig.1d). Namely, on day 37, the total nitrogen removal efficiency in the two systems was 9.33% and 36.13%, respectively. On day 63, the removal efficiency of total nitrogen was 35.63% in the control, while that in the ferrihydrite group reached 69.49%. This difference indicated that ferrihydrite effectively promoted nitrogen removal. Remarkably, the nitrogen removal efficiency in the control group was 9.33% on day 37 and rose to 35.63% on day 63. This was related to the presence of endogenous Fe(III) (49.85 mg/L) in the sludge, which was mainly originated from the sludge dewatering process that used iron salts as coagulants. NO2– and NO3- are also potential products of Feammox (eq. 2-3); however, these species were nearly undetected in this study (Fig.S2 and Fig.S3). One reason for this 11



result was that N2 was the dominant product of Feammox thermodynamically (eq. 1-3), which lowered the production of NOx-. Zhou et al. also found that NOx- was nearly undetected during the Feammox process in paddy soil.16 The N2 directly produced from Feammox was reported to account for 67% to 78% of the total N2 emission.11 On the other hand, the NOx– generated was easily reduced by organic matters (namely, denitrification) and Fe(II) (E(NO2-/N2O)= 1.31 V vs E(Fe3+/Fe2+) = 0.2 V, pH=7). Changes of iron. As a product of Feammox driven by dissimilatory Fe(III) reduction, the Fe(II) content could be used to quantify the extent of Fe(III) reduction. As shown in Fig.2a, on day 6, the Fe(II) content in the ferrihydrite group was 1026.78 mg/L, approximately ten-fold higher than that in the control group (106.98 mg/L). Besides more nitrogen removal in the ferrihydrite group (Fig.1d), TCOD removal efficiency reached 82.22%, compared with 48.52% in the control (Fig.3). Therefore, the generation of more Fe(II) was consistent with the more nitrogen and TCOD removal in the ferrihydrite group compared with the control. Fig.2b illustrates the changes in the Fe(III) content of the control. As shown in Fig.2a and Fig.2b, the contents of Fe(II) and Fe(III) fluctuated. In this anoxic system, the dissimilatory Fe(III) reduction by organic matters and NH4+ (i.e., Feammox) as electron donors was responsible for the generation of Fe(II). The absence of detectable NOx- in this study did not necessarily mean that no NOx- was produced. Instead, anaerobic NDFO (eq. 4-5) might explain the increase of Fe(III) and decrease of Fe(II) (Fig.2a and Fig.2b). 12


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However, 8 or 6 moles Fe(II) could be produced when producing 1 mole NO3- or NO2- according to eq. 2 and 3, while only 5 moles (eq. 4) and 2 moles (eq.5) Fe(III) could be produced when even all NO3- and NO2- were reduced to N2 via NDFO. The imbalance of electron transfer between Fe(III) reduction and Fe(II) oxidation might lead to less and less NOx- production in each circle, then gradually weakening NDFO and Fe(III) regeneration. This meant that the produced Fe(II) cannot be completely transformed back to Fe(III) when only considering Feammox and subsequent NDFO. Regarding the fluctuant changes of Fe(II) and Fe(III) content (Fig.2), it was assumed that other original metal oxides was involved in the N/Fe cycle. From EDX results (Fig.S4), besides iron, heavy metals Cr, Pd. et al., were detected in the sludge. These polyvalent metal oxides were reportedly capable of inducing dissimilatory metal reduction,29, 30 thereby likely participating to ammonium oxidation like Feammox,31-33 which is required to be further studied. If this assumption was true, the extra NOxmight be produced from ammonium oxidation by these polyvalent metal oxides, further oxidizing Fe(II). Similar results of the Fe(III) and Fe(II) during Feammox were also observed in sediment.16 10Fe(II) + 2NO3- + 12H+ → 10Fe(III) + N2 + 6H2O 4Fe(II) + 2NO2- + 8H+ → 4 Fe(III) + N2 + 4H2O

(4) (5)

Potential NDFO process. NDFO was possible in this study when considering that NOx- was the product of Feammox (eq. 2-3), particularly with the consumption of organics, denitrification gradually decreased and NDFO likely gradually increased. It means that Fe(III) is likely regenerated by NDFO in this study. To further identify the 13



iron transformation during NDFO, the NDFO experiment in the inoculants taken from the ferrihydrite-supplemented group was conducted. As shown in Fig.4a, the NO3--N content decreased rapidly from the initial level of 17.1 mg/L to 3.09 mg/L on day 2, resulting in a decrease in the Fe(II) content from the initial level of 51.32 mg/L to 10.98 mg/L on day 5, while the Fe(II) content of the NO3–_free group remained nearly unchanged (Fig.4b). Similar results also occurred in the reactor amended with NO2-, e.g., Fe(II) content also decreased sharply when supplementing NO2- (Fig.S5). This result indicated that NDFO occurred in the presence of NOx- and Fe(II) in this system, which was consistent with the results of Bao and Li,34 who found that Fe(II) was oxidized to Fe(III) when NO3- was added to an Fe(II)-containing anoxic system. Fe(II) cannot be oxidized to Fe(III) under anoxic conditions unless stronger oxidizing agents,35 e.g., NOx- are present (eq. 4-5). The NOx- species produced from Feammox likely served as an electron acceptor in the oxidation of Fe(II) through NDFO. In turn, the possible regenerated Fe(III) could likely participate in the Feammox process again, resulting in the further nitrogen removal. The further solid evidence supporting NDFO occurrence in this system will be explored in future studies. Iron speciation during the incubation. The physiochemical nature of the Fe (hydr)oxide, including Fe(III) reduction and possible NDFO, could influence Fe (hydr)oxide transformation.18 As demonstrated in Fig.5a, the XRD pattern of the original sludge showed characteristic peaks at 21.2°, 26.3°, 36.4° , 46.8°, 60.19° ascribed to the (1052), (2874), (772), (706) and (662) planes, corresponding to the standard card of iron hydroxide (Fe(OH)3) (JCPDS Card NO. 38-0032). It indicated 14


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that the endogenous Fe(III) in the original sludge from the dewatering process was in the form of iron hydroxide. The peaks located at 709.9 and 724.0 eV20 of binding energies shown in XPS pattern (Fig.5b) indicated the presence of Fe(II) and Fe(III), consistent with that Fe(II) and Fe(III) both existed in original sludge (Table 1). The Fe(III) added as ferrihydrite in ferrihydrite group was often inadequately described as Fe(OH)3 because these two iron compounds have similar physicochemical properties.36 A slight change in the XRD spectrum of the control group was observed after the experiment, i.e., besides iron hydroxide (Fe(OH)3) (Fig.6a), a new iron species, iron tartrate hydrate (C4H4FeO6·H2O) (Fig.6b), was formed. Specifically, the characteristic peaks at 21.2°, 26.3°, 36.4° ,60.19°, 67.92° ascribed to the (1002), (3728), (1057), (618) and (599) planes of iron hydroxide (JCPDS Card NO. 38-0032), and the characteristic peaks at 26.2°, 32.4°, 50.07° were ascribed to the (3728), (1006) and (889) planes, corresponding to the standard card of iron tartrate hydrate (JCPDS Card NO. 23-0299). While the XRD pattern of the iron minerals in the slurry of the ferrihydrite group after the experiment showed that the characteristic peaks at 30.09°, 35.42°, 37.05° were ascribed to the (1504), (1602) and (1602) planes, corresponding to the standard card of magnetite (JCPDS Card NO. 19-0629) (Fig.7a), and the characteristic peaks at 26.72°, 35.16°, 39.22° were ascribed to the (1438), (1326) and (1282) planes of akaganeite (β-FeOOH) (Fig.7b) (JCPDS Card NO. 34-1266), i.e., magnetite and akaganeite were formed after experiment. It meant that the original ferrihydrite transformed into magnetite and akaganeite. This result was consistent with those of previous studies,18 showing that ferrihydrite could be converted to 15



magnetite and other Fe(III) oxides. Besides possible NDFO, Boland et al. indicated that with the presence of aqueous Fe(II), ferrihydrite could be chemically transformed to more crystalline minerals.37 The observed changes in the crystalline iron, especially in the ferrihydrite group, indicated that the Fe(III) reduction and regeneration. Proportion of iron reduction associated with Feammox. Under anoxic conditions, the potential of assimilation by microbes, anammox, denitrification and possible NDFO could contribute to nitrogen loss (Table 2).7 However, for the assimilation, NH4+ could be converted by microbes to nitrogenous organic matters such as proteins, which might be mostly decomposed into NH4+ again. The reminder still stayed in the sludge slurry with no way to become N2 to generate nitrogen loss during assimilation. For denitrification, anammox and NDFO, these processes are relied on Feammox to generate NOx-. Therefore, the nitrogen loss could be resulted from the following two pathway: (i) N2 directly produced from Feammox; (ii) Feammox-generated NOxfollowed by denitrification, possible NDFO or anammox (anammox was ignored based on microbiological analysis below). In other words, the nitrogen loss in this study was caused by Feammox, regardless of directly producing N2 or firstly producing NOxthen being reduced to N2 (Fig.8). Organic materials and ammonium can both serve as electron donors in the dissimilatory Fe(III) reduction. Based on thermodynamics, the majority of Fe(III) reduction was involved preferentially with the oxidation of organics rather than with ammonium. Therefore, the proportion of Fe(II) generated from Feammox was not high. Ding et al. found that only 0.81-2.2% of the Fe(III) reduction in paddy soils was associated with Feammox, and increasing the organic content led to 16


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a decrease in the amount of Fe(II) generated from Feammox.11 The organic content in the wastewater treatment system in this study was much higher than that in the natural environment; thus, the proportion of Fe(II) generated from Feammox should be lower in this study than in that by Ding et al. In contrast, based on the nitrogen loss and the stoichiometric Fe/N ratio of 3 (eq. 1), the Fe(II) generated from Feammox in the ferrihydrite group accounted for 30.3% and 65.0% of the total Fe(II) on days 37 and 63, respectively, which were much higher than the proportions reported in previous studies (0.81-2.2% and 0.4−6.1%).11, 16 It was likely because that NDFO was indispensable to re-produce Fe(III) (Fig.8), consistent with NOx--reducing Fe(II) oxidizer (see Microbial community below) detected in systems, leading to the more nitrogen removal and higher proportion of Fe(III) reduction through Feammox. Notably, the endogenous Fe(III) content of 49.85 mg/L in the control was not sufficient for removing such a large amount of nitrogen by day 63. Specifically, only 4.15 mg/L of nitrogen could theoretically be removed via Feammox based on the 49.85 mg/L Fe(III) content in the control based on the stoichiometric Fe/N ratio of 3 (eq. 1). Nevertheless, 37.94 mg of nitrogen was removed on day 63 in the control, which was more than the theoretical amount, implying that polyvalent metal oxides existing in the sludge (Fig.S4) was likely involved in the nitrogen removal. Importantly, the operating time was another factor that influences the proportion of Fe(III) reduction associated with Feammox. In general, the proportion of Fe(III) reduction associated with Feammox in the ferrihydrite group increased over time, e.g., from 30.3% on day 37 to 65.0% on day 63. As the organic matters were depleted during digestion, ammonium gradually 17



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became the main electron donor for Fe(III) reduction. Microbial community. To date, the underlying microbiological process of Feammox has not been clarified. Most researchers have suggested that iron-reducing bacteria participate in Feammox (Fig.S6). For example, Zhou et al. ascribed the Feammox process in paddy soil to the function of Geobacter, an iron-reducing bacterium.16 Huang and Jaffé stated that an uncultured Acidimicrobiaceae bacterium capable of reducing Fe(III) played a key role in Feammox in a forested riparian wetland.27 In this study, the iron-reducing bacteria were also enriched. As shown in Fig.9a, Deltaproteobacteria, which contains iron-reducing bacteria, e.g., Geobacter, accounted for 6.02% of the bacteria 16S rRNA gene sequences at the class level in the control,

while this class accounted for 12.8% of the bacteria in the

ferrihydrite-supplemented group. Further identification at the genus level showed that Geobacter made up 0.04% and 0.48% of the total sequences in the control and ferrihydrite groups, respectively (Fig.9b). The low abundance of Geobacter in the sludge agreed with the results of previous reports,38 indicating that the inoculant sludge was not an ideal system for enriching Geobacter compared with the natural habitat. This low abundance of Geobacter was probably related to the low efficiency of nitrogen removal in this study (69.49% on day 63 in the ferrihydrite group, Fig.1d). The

NO3--reducing

Fe(II)

oxidizer

Acidovorax

(Fig.9b)

and

the

family

Comamonadaceae (Fig.9c), belonging to iron-oxidizing bacteria, were also detected in both groups. Particularly, the abundance of Acidovorax was 1.18% and 0.81% in the control and ferrihydrite groups, respectively, and the abundance of 18


Page 19 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Comamonadaceae

was

1.28% and

0.44%.

Putative

denitrifiers,

including

Acinetobacter, Rhodoplanes, Bacillus and Pseudomonas,39 were detected in the two groups, indicating that denitrification of NOx- from Feammox was an alternative to the consumption of NOx- (Fig.S2 and Fig.S3). Notably, one microbe at the genus level, Aminicenantes_genera_incertae_sedis, was abundantly enriched in the control (19.88%) and the ferrihydrite group (40.34%) (Fig.9b). Aminicenantes has been found to adapt and utilize a variety of complex sugar polymers and amino acids, such as glycine, glutamate and aspartate,40 which was consistent with the observed polysaccharide and protein removal (Fig.1b and Fig.S7). The genes for the assimilatory acquisition of nitrogen were also identified in Aminicenantes. However, the ability of Aminicenantes_genera_incertae_sedis to reduce Fe(III), even when mediated by Feammox, is unknown.

IMPLICATION Ammonium is one of mainly concerned pollutants due to the risk to cause eutrophication. Feammox has increasingly been reported in natural systems. However, this nitrogen removal model has rarely been investigated in wastewater treatment system. In this study, the nitrogen removal efficiency in the ferrihydrite group was significantly higher than that of the control (Fig.1d), implying that ferrihydrite induced anaerobic NH4+ oxidation and further nitrogen removal. Although the nitrogen removal efficiency was not high, e.g., 69.49% of nitrogen was removed during 63 days when supplementing ferrihydrite, the study still demonstrated the 19



possibility of application of Feammox in anaerobic nitrogen removal from wastewater. Although the results suggested that Feammox was related to nitrogen removal, the direct evidence still lacked.

15

N-labeled isotopic is an ideal tool to characterize

nitrogen loss in Feammox, which will be investigated in our next study. CONCLUSIONS In this study, ferrihydrite was supplemented in anaerobic wastewater treatment reactor to investigate its effects on nitrogen removal. The results showed that nitrogen removal efficiency increased by 33.86% after 63 days compared to the control. As part products of Feammox, Fe(II) and NOx- likely reacted together in this study, resulting in NDFO to generate Fe(III). XRD results showed that new species of Fe(III) compounds, magnetite and akaganeite, were detected during Fe (Hydr)oxide transformation. Microbial community analysis demonstrated that iron-reducing bacteria and iron-oxidizing bacteria were enriched in the two groups, indicating the cooperation of two bacteria was essential for the ammonium removal in Fe(III)-containing wastewater treatment systems.

ASSOCIATED CONTENT Supporting Information

The morphology of the synthesized ferrihydrite via an scanning electron microscope; Specific method of Fe(II) and Fe(III) determination; DNA extraction, PCR amplification and high-throughput 16S rRNA pyrosequencing; NO3- content sometimes during experiment; NO2- content sometimes during experiment; EDX of sludge slurry taken from a sludge treatment plant; Changes in Fe(II) contents when with 20


Page 20 of 35

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and without NO2- under anoxic conditions; Mechanism model of Feammox; Total polysaccharide content in different groups on day 37.

AUTHOR INFORMATION Corresponding Author * Tel: +86-411-84706140. Fax: +86-411-84706263. E-mail: [email protected]. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Scientific Foundation of China (21777016).

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27



Page 28 of 35

Table Table 1. Main initial characteristics before incubation. a

a b

Parameters

Initial value

TCOD b

4515±1.62 mg/L

Total Polysaccharide

306.56±11.69 mg/L

Total protein

191.87±14.20 mg/L

NH4+-N

10.14±0.51 mg/L

NO3--N

Undetected

NO2--N

Undetected

Total nitrogen

106.48±12.65 mg/L

Fe(II)

72.87±13.35 mg/L

Fe(III)

49.85±7.17 mg/L

Average data and standard deviation obtained from triplicate tests. TCOD: total chemical oxygen demand.

Table 2. Potential pathway of nitrogen loss under anoxic conditions

a

N loss

mechanism

Feammox

Eq.1-3

anammox

NH4+ + NO2- → N2 + 2H2O

assimilation by microbes

Used for microbial proliferation.

denitrification

Organics act as electron donors

NDFOa

Eq. 4 and 5

NOx--dependent Fe(II) oxidation.

28


Page 29 of 35

Figure

75

+ NH4 -N content (mg/L)

+ 4

60

NH was added

NaHCO3 was added

60

45 45

30

15

(b) 200

100

150

Protein concentration Removal efficiency

100

80

60

50

40

0

20

Removal efficiency(%)

Ferrihydrite

Protein content (mg/L)

Control Ferrihydrite

+ NH4 -N content (mg/L)

(a) 75

30

0

20

40

60

80

100

Initial

120

Control

Ferrihydrite

Time (d)

(d)

Efficiency of control Efficiency of ferrihydrite Control Ferrihydrite

Total nitrogen content (mg/L)

120

12

9

6

3

90

100

80

60 60

40

20

30

0 0

0 0

5

10

15

20

25

30

Initial

Day 37

Day 63

Time(d)

Fig.1. (a) Changes in the ammonium content in the control and ferrihydrite groups during the experiment; (b) total protein content in the different groups on day 37; (c) changes in the ammonium content under abiotic conditions; and (d) total nitrogen and removal efficiency on days 37 and 63 for the two groups. Error bars represent standard deviations.

29


Removal efficiency (%)

(c) 15 + NH4 - N content (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2000

1600

Page 30 of 35

(b) 160

Control Ferrihydrite

Ferric iron content (mg/L)

(a)

1200

800

400

0

Control

120 80 40 0 -40 -80

0

15

30

45

60

0

15

30

45

Time (d)

Time (d)

Fig.2. (a) Fe(II) contents in the different groups during the experiment and (b) Fe(III) contents in the control group during the experiment. Error bars represent standard deviations.

5000

100

Removal efficiency

4000

80 3000 60 2000 40

Removal efficiency(%)

TCOD content

TCOD content (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ferrous iron content (mg/L)


1000

0

20

Initial

Control

Ferrihydrite

Fig.3. TCOD contents in the different groups on day 37. Error bars represent standard deviations.

30


60

Page 31 of 35

(a)18

(b)

-

15

Ferrous iron content (mg/L)

with NO3

NO3 -N content (mg/L)

-

without NO3

12

-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9 6 3 0

75

with NO3

-

without NO3

60 45 30 15 0

-3 0

6

12

18

24

0

30

6

12

18

24

30

Time (d)

Time (d)

Fig.4. Changes in the (a) NO3- and (b) Fe(II) contents when with and without NO3under anaerobic conditions.

Fig.5. XRD (a) and Fe2p XPS (b) spectrum patterns of the original sludge. The XRD pattern for standard rutile is shown as straight lines (a).

31



(b)

(3728)

(3728)

Intensity

Intensity

(a)

(1002)

(1006)

(889)

(1057) (618) (599)

iron hydroxide

20

40

60

80

Iron tartrate hydrate 20

100

40

60

80

100

2-theta (degree)

2-theta (degree)

Fig.6. XRD pattern of the iron minerals in the sludge of the control group after the experiment. The XRD pattern for standard rutile is shown as straight lines [iron hydroxide (a) and iron tartrate hydrate (b)].

(a)

Intensity

(1282)

(1326)

(1438)

(1602)

(1504)

Intensity

(b) (1602)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

magnetite

20

40

60

80

akaganeite

100

20

40

60

80

100

2-theta (degree)

2-theta (degree)

Fig.7. XRD pattern of the iron minerals in the sludge of the ferrihydrite group after the experiment. The XRD pattern for standard rutile is shown as straight lines [magnetite (a) and akaganeite (b)].

32


Page 33 of 35

Fig.8. The diagram of nitrogen removal in this study.

(a) 100 Relative abundance(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Others Methanomicrobia Thermotogae Sphingobacteriia Caldilineae Synergistia Betaproteobacteria Planctomycetia Actinobacteria Bacilli Clostridia Alphaproteobacteria Gammaproteobacteria Deltaproteobacteria Bacteroidia Anaerolineae unclassified

80

60

40

20

0

Control

Ferrihydrite

33



Relative abundance (%)

(b)

100

Others Geobacter Pseudomonas Litorilinea Syntrophomonas Acidovorax Olsenella Thermogutta Syntrophorhabdus Citrobacter Bellilinea Ornatilinea Smithella Exiguobacterium Levilinea Longilinea Mariniphaga unclassified Aminicenantes_genera _incertae_sedis

80

60

40

20

0

Control

Ferrihydrite

(c) 100 Relative abundance(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Others Saprospiraceae Geobacteraceae Hyphomicrobiaceae Pseudomonadaceae Marinilabiliaceae Comamonadaceae Rhodobacteraceae Syntrophomonadaceae Moraxellaceae Coriobacteriaceae Syntrophorhabdaceae Caldilineaceae Synergistaceae Enterobacteriaceae Planctomycetaceae Bacillales_Incertae Sedis XII Syntrophaceae Prolixibacteraceae Anaerolineaceae unclassified

80

60

40

20

0

Control

Ferrihydrite

Fig.9. Relative abundances of bacteria at the class level (a), genus level (b) and family level (c) in the different groups on day 87.

34


Page 34 of 35

Page 35 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic:

Feammox, i.e., anaerobic ammonium oxidation coupled to Fe(III) reduction, plays an important role in the Fe/N cycle.

35


Transformation of Nitrogen and Iron Species during Nitrogen Removal

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