Performance of nitrate-dependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitrification

Performance of nitrate-dependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitrification

Accepted Manuscript Performance of nitrate-dependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitr...

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Accepted Manuscript Performance of nitrate-dependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitrification Meng Zhang, Ping Zheng, Wei Li, Ru Wang, Shuang Ding, Ghulam Abbas PII: DOI: Reference:

S0960-8524(14)01773-8 http://dx.doi.org/10.1016/j.biortech.2014.12.036 BITE 14358

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

13 October 2014 9 December 2014 11 December 2014

Please cite this article as: Zhang, M., Zheng, P., Li, W., Wang, R., Ding, S., Abbas, G., Performance of nitratedependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitrification, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.036

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Performance of nitrate-dependent anaerobic ferrous oxidizing (NAFO) process: A novel prospective technology for autotrophic denitrification Meng Zhang a, Ping Zheng a,*, Wei Li a, Ru Wang a, Shuang Ding a, Ghulam Abbas a a

Department of Environmental Engineering, Zhejiang University, Hangzhou 310058,

China

*Corresponding author: Ping Zheng Department of Environmental Engineering, Zhejiang University Hangzhou 310058, China Tel: 0086 517 88982819 Fax: 0086 571 88982819 E-mail: [email protected]

1

Abstract: Nitrate-dependent anaerobic ferrous oxidizing (NAFO) is a valuable biological process, which utilizes ferrous iron to convert nitrate into nitrogen gas, removing nitrogen from wastewater. In this work, the performance of NAFO process was investigated as a nitrate removal technology. The results showed that NAFO system was feasible for autotrophic denitrification. The volumetric loading rate (VLR) and volumetric removal rate (VRR) under steady state were 0.159 ± 0.01 kg-N/(m3•d) and 0.073 ± 0.01 kg-N/(m3•d), respectively. In NAFO system, the effluent pH was suggested as an indicator which demonstrated a good correlation with nitrogen removal. The nitrate concentration was preferred to be less than 130 mg-N/L. Organic matters had little influence on NAFO performance. Abundant iron compounds were revealed to accumulate in NAFO sludge with peak value of 51.73% (wt), and they could be recycled for phosphorus removal, with capacity of 16.57 mg-P/g VS and removal rate of 94.77 ± 2.97%, respectively. Keywords: autotrophic denitrification, NAFO, working performance, operation conditions, process mechanism

1. Introduction Nitrate contamination of surface water and ground water is an increasingly serious problem around the world, which could lead to multidimensional problems, e.g., eutrophication, infant methemoglobinemia, and serious disease such as cancer. The nitrate could originate from nitrate containing wastewaters, such as landfill leachate, chemical fertilizers, and industrial/domestic wastewaters, and it could also come from 2

the nitrification process (ammonium oxidation to nitrate) in wastewaters treatment plants. (Chen et al., 2014; Chung et al., 2014; Manconi et al., 2007; Qu and Fan, 2010). Biological denitrification process is a well developed and widely applied approach to fight nitrate pollution. It can be divided into heterotrophic and autotrophic processes. Heterotrophic process is efficient in the presence of sufficient carbon source. However, the supply of carbon source often caused a secondary pollution as well as a high cost (Chung et al., 2014; Zhang and Lampe, 1999). Autotrophic process has attracted wider attention due to its obvious advantages of less operation cost, lower sludge production, and better applicability to the wastewater with low C/N ratio (Ghafari et al., 2008; Henze et al., 2002; Park and Yoo, 2009). Nitrate-dependent anaerobic ferrous oxidation (NAFO) is a process using ferrous iron as electron donor to convert nitrate to nitrogen gas under anoxic conditions (Straub et al., 1996; Weber et al., 2006). This is an autotrophic process yielding energy, since the redox potential of ferrous/ferric couples is about +200 mV and it is lower than that of nitrate/nitrogen gas (+710 mV) in circum-neutral environment (Hedrich et al., 2011). In 1996, NAFO microorganism was reported by Straub et al. (1996) for the first time, and since then extensive research works were conducted (Hedrich et al., 2011; IIbert and Bonnefoy, 2013; Weber et al., 2006). So far, NAFO process was detected in various habitats, such as lake sediments, freshwater and swine waste lagoon (Chaudhuri et al., 2001; Muehe et al., 2009; Straub et al., 1996, 2004). These 3

environments are characterized by Fe(II) that comes from an anoxic source with the supply of nitrate from a nitrate-containing water body. NAFO bacteria are assumed to participate in the biogeochemical cycles of iron and nitrogen in these habitats (Fig. S1) (Wang et al., 2003; Clément et al., 2005; Weber et al., 2006a, b ; Zhang et al., 2014). Based on the widespread NAFO, a novel autotrophic denitrification technology was developed, whose product (ferric salts) was proposed to be used in phosphate removal from wastewaters (Hao et al., 2011; Stone, 2011; Zhang et al., 2014). Although NAFO process was discovered about 20 years ago, scarce information is available on the application of NAFO process, especially in the field of wastewater treatment. So the working performance and operation conditions of NAFO process as an autotrophic denitrification technology were investigated using an up-flow anaerobic sludge bed (UASB) reactor. A comparative analysis between seeding sludge and NAFO sludge was conducted to demonstrate the characteristics of NAFO sludge. The feasibility of this technology for phosphate removal was finally evaluated to recycle the iron salt in NAFO sludge.

2. Materials and methods 2.1. Seeding sludge and synthetic wastewater The reactor was inoculated with anaerobic granular sludge taken from a full-scale UASB reactor treating wastewater from paper mill. The total solid (TS) and volatile solids (VS) of the seeding sludge were 140.2 ± 1.8 g/L and 61.10 ± 0.5 g/L, respectively. Synthetic wastewater was supplied to investigate the working 4

performance of NAFO process, and the components were as follows (g/L): NaHCO3 2.5, MgSO4·7H2O 0.5, CaCl2·2H2O 0.01, (NH4)2SO4 0.28, KH2PO4 0.25, and trace element solution (1 ml/L). The trace element solution contained (Kumaraswamy et al., 2006; Wang et al., 2014; Weber et al., 2009) (g/L): EDTA 3.0, CoCl2·6H2O 0.19, MnCl2·2H2O 0.50, ZnCl2 0.07, H3BO3 0.01, NiCl3·6H2O 0.024, CuCl2·2H2O 0.01, Na2MoO4·2H2O 0.036. Nitrate and ferrous iron were supplied to mineral medium as required in the form of NaNO3 and FeSO4•7H2O, respectively.

2.2. NAFO bioreactor The NAFO process was investigated through a UASB reactor made of plexiglass as shown in Fig. S2. The configuration parameters of the reactor were: internal diameter Φ 65 mm, total height 700 mm, and total working volume 0.8 L. The synthetic wastewater was prepared in a 1.5 L sealed glass bottle. The initial pH (7.0 or so) was regulated by NaOH with a concentration of 2.0 mol/L. The synthetic wastewater was pumped into the bottom of the reactor, and the synthetic medium moved gradually upward through the granular sludge. After a solid-liquid separation, the treated wastewater flowed out of reactor. The temperature was controlled at 30 ± 1℃ during the experiment.

2.3. Jar test for phosphorus removal with NAFO sludge Through the jar test, the phosphorus removal experiment was carried out using NAFO sludge. NAFO sludge was washed three times with 0.9% NaCl solution before 5

experiments. In each test, 50 ml of synthetic wastewater with initial pH of 7.0 ± 0.1 was taken into a 65 ml serum bottle, and then it was purged with Ar gas for 10 min to displace the headspace air. Then, 5 ml washed sludge was added to the system. After shaking at speed of 150 r/min for 0.5 h and 1.5 h at 25±1℃, the supernatants were collected through a membrane filter with a nominal pore diameter of 0.45 µm. The PO43-, Fe2+ and total iron concentrations were immediately determined, respectively. Experiments with seeding sludge and without sludge were also set as control. All the serum bottles were sealed with butyl rubber stoppers and aluminum caps. All the tests were run in triplicates.

2.4. Characteristics of granular sludge At first, the morphological characteristics of anaerobic granular samples (inoculum) and NAFO granular samples were observed with digital camera (Canon IXUS 1100HS, Japan) and a stereoscope Discovery V8 (ZEISS, Germany) (Wang et al., 2014). Then, the samples were observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for detailed examination, using chemical fixation (SEM and TEM) and epon coating (TEM). The procedures of SEM and TEM were followed as reported by Tang et al. (2010). The SEM and TEM imaging was performed with the model SIRON (FEI, Holland) and JEM-1200EX (NEC, Japan). The elemental contents in anaerobic granular samples (inoculum) and NAFO granular samples were also analyzed using energy-dispersive X-ray spectroscopy 6

(EDS).

2.5. Analytical methods The samples were filtered through membranes (0.45 µm pore diameter) and analyzed immediately after collection. The determination of NO3--N, NO2--N, Fe2+, total iron and PO43- was performed according to the standard methods (APHA, 1998). The NO3--N concentration was measured by the ultraviolet spectrophotometric screening method.

The

NO2--N

concentration

was

measured

colorimetriclly

using

N-(l-naphthyl)-ethylenediamine dihydrochloride. The ferrous iron and total iron concentrations were measured colorimetrically using 1,10-phenanthroline. The PO43concentration was measured by the ascorbic acid photometric method. The temperature, total solids (TS) and volatile solids (VS) were measured following the standard methods (APHA, 1998). The pH was determined by a S20 K pH meter (Mettler Toledo, Switzerland).

3. Results and discussion 3.1. Performance of nitrate removal by NAFO process Continuous experiment was carried out with a UASB reactor to investigate the working performance of NAFO process. The results are shown in Fig. 1. As depicted in Fig. 1-A, the hydraulic retention time (HRT) was shortened from 18.75 h to 18 h during the first 22 d, and the influent concentrations of nitrate and ferrous iron were increased from 32.29 mg-N/L and 313.24 mg-Fe/L to 139.55 mg-N/L and 1703.4 7

mg-Fe/L, respectively. The corresponding nitrate and ferrous iron removals were high (over 95%) with residual concentrations of 0-0.12 mg-N/L and 0-72 mg-Fe/L, respectively. During 22-32 d, the HRT was shortened to 17.25 h. The influent concentrations of nitrate and ferrous iron were decreased to 113.96 ± 5.64 mg-N/L and 1491.75 ± 101.51 mg-Fe/L. The increase in corresponding concentrations of residual nitrate and ferrous iron were 60.82 ± 4.53 mg-N/L and 929.91± 123.72 mg-Fe/L, respectively. The NAFO process was observed at its steady state during 32-47 d with nitrate and ferrous iron removals of 45.63 ± 3.31% and 43.94 ± 3.55%, respectively. Throughout the operation of reactor, the concentration of nitrite (intermediate of nitrate reduction) was less than 1 mg-N/L. As evident from Fig. 1-A, a positive correlation was noticed between nitrate and ferrous iron conversion. During the experiment, the average concentration of nitrate and ferrous iron in the influent and effluent were 86.51 mg-N/L, 1114.45 mg-Fe/L and 24.30 mg-N/L and 389.76 mg-Fe/L, respectively. So, the consumed Fe/N molar ratio was calculated to be 2.91, which was in accordance with literature values(Fe/N = 1.28- 5.23) (Blöthe and Roden, 2009; Li et al., 2014; Nielsen and Nielsen, 1998). Fig. 1-B shows the volumetric capacity of nitrogen removal by NAFO process. The maximum VRR of nitrate in this study was 0.186 kg-N/(m3•d) and it was 0.073 ± 0.01 kg-N/(m3•d) during the steady state. However, according to Nielsen et al. (1998), the nitrate conversion activity of activated sludge in NAFO process could be achieved was 0.31 mmol N/(gVSS • h), demonstrating that a potential VRR by NAFO process could be as high in this work as 4.42 kg-N/(m3•d). Compared with other autotrophic 8

denitrification technologies, such as sulphur-dependent denitrification (0.05-3.11 kg-N/(m3•d)), hydrogen-dependent denitrification (0.027-2.419 kg-N/(m3•d)) and bio-film-electrode reactor (0.034-0.227 kg-N/(m3•d)) (Karanasios et al., 2010; Park and Yoo, 2009; Show et al., 2013; Zhao et al., 2011), the performance of NAFO process was fairly high, and it deserves to be developed further. Insert Fig. 1

3.2. Operation conditions for nitrate removal by NAFO process 3.2.1. Effluent pH Fig. 2 shows the pH effect on nitrate removal in NAFO process. The pH in the influent was set at 6.63 ± 0.11 during the 74d operation period. The pH in the effluent, however, varied significantly in the range of 3.01-7.22. The performance of nitrogen removal showed a similar trend when the effluent pH decreased progressively during this period. Fig. 2-B illustrates a positive relationship between effluent pH and nitrate removal with a linear correlation coefficient (R2) of 0.8444, suggesting that effluent pH could be an indicator for nitrogen removal in NAFO system. Hence, a feedback regulation of pH value through effluent was preferred to control NAFO process. It should be noted that the percentage of nitrogen removal was above 95% as long as the effluent pH value was over 6.0. According to Equation 1 (Straub et al., 1996; Weber et al., 2006), the NAFO process would acidify the reaction system through producing hydrogen ions. Higher pH would neutralize the reaction and strengthen the forward reaction according to Le Châtelier’s Principle (Petrucci et al., 2004). On the other 9

hand, circum-neutral environment would favor ferrous iron to act as electron donor to yield energy since the redox potential of ferrous/ferric couple(s) at circum-neutral (and higher) pH is much lower (about +200 mV) than that in acidic liquors (about +770 mV) (Hedrich et al., 2011; Widdel et al., 1993). 10FeCO3 + 2NO3- + 24H2O → 10Fe(OH)3 + N2 + 10HCO3- + 8H+

(1)

Insert Fig. 2

3.2.2. Nitrate concentration According to the rate law, substrate concentration is an important factor affecting reaction rate (Petrucci et al., 2004). Table 1 lists the influence of nitrate concentration on NAFO process. When nitrate concentration went up from 32.13±0.43 mg-N/L to 173.54±1.96 mg-N/L, the VLR increased from 0.041 kg-N/ (m3•d) to 0.252 kg-N/ (m3•d), with a decline of nitrate removal efficiency from 100% to 35.28%. The VRR showed a downward trend after the first rise with a peak value of 0.136 kg-N/ (m3•d) at nitrate concentration of 127.23±1.14 mg/L. Hence, a proper nitrate concentration with values less than 130 mg-N/L is suggested for NAFO process. Insert Table 1

3.2.3. Organic matter Usually, organic matters serve as carbon source for heterotrophic organisms. The presence of organic matter will enrich the heterotrophic denitrification bacteria, and weaken the autotrophic denitification (Park and Yoo, 2009; Qu and Fan, 2010; 10

Rittmann and McCarty, 2002). However, table S1 shows a different result when acetate was added as organic matter to NAFO system. With influent nitrate and organic matter concentrations of 102.02 ± 3.18 mg-N/L and 203.83 ± 3.72 mg/L (COD), respectively, the nitrate removal did not fluctuate significantly within 72 hours of batch experiments. The relative standard error was only 0.13%. This result revealed a good resistance of NAFO system to organic matter.

3.3. Characteristics of NAFO sludge 3.3.1. VS/TS value Activated sludge serves as a biocatalyst and plays a significant role in NAFO process. For sludge characteristics, VS/TS is one of the important indicators. The VS/TS values of seeding sludge and NAFO sludge were determined. The results showed that the TS of NAFO sludge increased significantly from 140.2 ± 1.8 g/L to 314.8 ± 11.4 g/L, and the VS decreased slightly from 61.10 ± 0.5 g/L to 50.65 ± 2.2. As a consequence, the VS/TS ratio dropped to 0.16 in NAFO sludge compared with 0.44 in the seeding sludge. In other words, after the operation, the content of organic matters in NAFO sludge decreased while that of inorganic matters increased in NAFO sludge.

3.3.2. Morphology and structure characteristics of sludge The sludge characteristics, such as morphology and structure were observed using digital camera, stereoscope, SEM and TEM to clarify the VS/TS changes in the sludge. After a long-term operation, the NAFO sludge aggregated and formed a rougher surface. The colors changed from black to yellow either in the surface or interior, as 11

shown in Fig. S3-a,b and Fig. S3-A,B. The amount of microorganisms in NAFO sludge declined slightly. Meanwhile, tiny particles around the cells increased as indicated by the SEM and TEM images. The microbial cells were covered by precipitates with crystalline, needlelike structures on the surface, which was reported as unique properties of NAFO biomass (Hohmann et al., 2010; Schädler et al., 2009) (Fig. S3-c,d and Fig. S3-C,D). The presence of tiny precipitates around the cells or covered on the surface was in accordance with the promotion of NAFO activity. Some certain relationship between the precipitates and NAFO activity was supposed to exist. EDS was used to analyze the elemental contents in the sludge. The results in Fig.S4 showed that, the iron content in NAFO sludge reached a peak value of 51.73% (wt), which was remarkably higher than that in the seeding sludge of 1.49% (wt). This indicated that iron compounds were the main component of precipitates in NAFO sludge, which should also be responsible for the decrease of VS/TS to 3.3.1. Furthermore, the iron compounds accumulated around or on the surface of cells could be either the substrate (ferrous iron) or product (ferric iron) of NAFO process. Direct contact between bacterial cells and ferrous iron may be a prerequisite for the reaction to take place. The production of ferric iron may also have a site preference for accumulation. Molecular techniques, such as qPCR and mRNA-based qPCR, and research on isolated iron-oxidizing bacteria may be helpful to reveal the mechanisms in the future study.

12

3.3.3. Characteristics of NAFO sludge for phosphorus removal In this work, NAFO system produced sludge with high content of iron compounds, which is an engineering drawback that could not be ignored in field applications of the NAFO. On the other hand, iron compounds were thought to be a potential precipitant for the control of phosphorus pollution, which was another widespread and challenging environmental problem nowadays (Camargo et al., 2005; Hao et al., 2011; Nguyen et al., 2014). Herein, jar test was carried out to test the feasibility of phosphorus removal by NAFO sludge. When the initial phosphorus concentration and pH were fixed at 87.44 ± 0.87 mg-P/L and 7.0 ± 0.1 respectively, the phosphorus removal efficiency reached 58.58 ± 1.74% after 0.5 h with the addition of 10% (v/v) NAFO sludge, and 94.77 ± 2.97% after 1.5 h. The corresponding VRR went up to 1.33 kg-P/(m3•d) (16.57 mg-P/g VS), which was a high level compared with value reported in literature (Barca et al., 2012; Nguyen et al., 2014; Zhang et al., 2014). The results demonstrated that the iron compounds accumulated in NAFO sludge and they could be recycled for phosphorus removal.

4. Conclusions The NAFO process was proved to be capable of autotrophic denitrification with a volumetric loading rate (VLR) of 0.159 ± 0.01 kg-N/(m3•d) and volumetric removal rate (VRR) of 0.073 ± 0.01 kg-N/(m3•d), respectively. The effluent pH was suggested as an indicator for nitrogen removal. The nitrate concentration was preferred to be less than 130 mg-N/L. Organic matters showed little influence on NAFO performance. 13

The iron compounds accumulated in NAFO sludge reached 51.73% (wt), and they could be recycled for phosphorus removal with capacity of 16.57 mg-P/g VS, removal rate of 94.77 ± 2.97% and VRR of 1.33 kg-P/(m3•d), respectively.

Acknowledgments This work was financially supported by Natural Science Foundation of China (No. 51278457), National Key Technology R&D Program (No. 2013BAD21B04), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20110101110078) and Zhejiang Key Science and Technology Innovation Team Project Grant (2013TDXX).

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Fig. 1 influent nitrate effluent nitrate

A

influent ferrous effluent ferrous

influent nitrite effluent nitrite

2000 1800 1600

120

1400

100

1200 80

1000 800

60

600 40 400 20

Iron concentration (mg/L)

Nitrogen concentration (mg/L)

140

200 0

0 0

5

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B

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HRT

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18 0.12 0.10 17

HRT (h)

3 Nitrogen removal rate (kg-N/(m .d))

0.18

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40

15 50

Time (d)

Fig. 1 - Performance of nitrate removal by NAFO process. A and B refer to substrate conversion and volumetric capacity, respectively.

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Fig. 2 A7

100

5

60

4

40

pH

80

influent pH effluent pH nitrogen removal

3

20

2 0

10

20

Nitrogen removal (%)

6

30

40

50

60

0 80

70

Time (d)

B 100 2

R =0.8444 Nitrogen removal (%)

80

60

40

20

0 3

4

5

6

7

Effluent pH

Fig. 2 - Effect of effluent pH on nitrate removal by NAFO process. A refers to changes along the operation. B refers to the linear correlation.

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Table 1 - Performance of NAFO process under different initial nitrate concentrations Nitrate concentration (mg-N/L) Removal (%)

VLR (kg-N/(m3· d))

VRR (kg-N /(m3·d))

Influent

Effluent

32.13±0.43

0.00±0.00

100.00

0.041

0.041

46.59±1.38

0.00±0.00

100.00

0.060

0.060

60.92±1.23

0.09±0.05

99.85

0.078

0.078

87.69±1.57

0.12±0.04

99.86

0.117

0.117

115.60±4.05

17.42±1.41

84.93

0.154

0.131

127.23±1.14

33.62±0.28

73.58

0.185

0.136

156.25±3.74

83.85±2.15

46.34

0.227

0.105

173.54±1.96

112.32±6.46

35.28

0.252

0.089

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Graphical abstract

Graphical abstract

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Highlights A novel NAFO system was experimentally proved viable for autotrophic denitrification. The nitrate volumetric removal rate was 0.073 ± 0.01 kg-N/(m3•d) by NAFO system. The iron content in NAFO sludge was high enough to be recycled for phosphate removal. NAFO system had the potential to remove nitrate and phosphorus simultaneously.

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