Fast start-up of Anammox process with appropriate ferrous iron concentration

Bioresource Technology 170 (2014) 506–512

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Fast start-up of Anammox process with appropriate ferrous iron concentration Zhen Bi a, Sen Qiao a,⇑, Jiti Zhou a, Xin Tang a, Jie Zhang b a Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, PR China b State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, PR China

h i g h l i g h t s 2+

 The start-up time of Anammox process could be shortened to 50 days with 0.09 mM Fe .  An increase of heme c content was the key for fast start-up of Anammox process. 2+

 The hydrazine dehydrogenase activity is highly relevant to increasing Fe

a r t i c l e

i n f o

Article history: Received 17 June 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 10 August 2014 Keywords: Anammox Start-up Fe effects Heme c Enzyme activity

a b s t r a c t In this study, three upflow column reactors were compared for anaerobic ammonium oxidation (Anammox) process start-up time with different ferrous iron concentration in feeding. Continuous experiments indicated that the start-up time of Anammox process could be shortened from 70 to 58 d in R2 (0.06 mM Fe2+) and 50 d in R3 (0.09 mM Fe2+). The Anammox activity appeared after 16 days operation in R3. Quantitative PCR (q-PCR) analysis demonstrated a significant increase in quantity of Anammox bacteria in R3 compared with the other two reactors during entire operation. At the Fe(II) concentration of 0.09 mM, the heme c levels inside Anammox cell and hydrazine dehydrogenase (HDH) activity increased dramatically, which could be the trigger of fast Anammox start-up. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The Anammox process has been put forward as a new and promising way to treat wastewater containing a high ammonium concentration and low COD content in the last years. Discovered in the middle of 1990s, Anammox process is one of the latest additions to the biological nitrogen cycle. In Anammox process, ammonium is anaerobically oxidized to N2 using nitrite as the electronic acceptors. This autotrophic process uses CO2 as the only carbon source. Compared with the conventional biological processes (nitrification–denitrification), Anammox process reduces aeration by 64%, exogenous electron donor by 100% and sludge production by 80–90% (van Loosdrecht et al., 2008). As a result, the Anammox process can save up to 90% of operation cost as compared to traditional nitrogen treatment processes (Jetten et al., 2001). However, Anammox organisms grow very slowly, with approximate doubling time of 7–20 d (Van der Star et al., 2007; Strous

⇑ Corresponding author. http://dx.doi.org/10.1016/j.biortech.2014.07.106 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

dosage.

et al., 1998; Kartal et al., 2012). For example, the start-up period of a full-size plant built in Rotterdam was approximately 2 years (Kuenen, 2008). Apparently, the start-up of Anammox reactor from conventional activated sludge became very difficult. During the past years, efforts to shorten start-up period have generally aimed at increasing the biomass retention of the reactor via selecting suitable reactor types (Jin et al., 2008; Tao et al., 2012), using sludge washout strategy (Kieling et al., 2007), and using different types of carriers (Wang et al., 2013; Chen et al., 2012). Apart from these approaches, a promising alternative to avoid the disadvantage of long start-up time is to promote the metabolism of Anammox biomass via enhancing the ammonia oxidation activity. By this way, the growth rate of Anammox bacteria could be accelerated, and then fast start-up of Anammox reactor could be achieved. Anammox bacteria rely on a specific set of heme proteins, such as hydrazine synthesis (HZS), hydrazine dehydrogenase (HDH), etc., for their metabolism. HZS is one of the most intriguing properties of Anammox which catalyze hydrazine production from ammonia and nitric oxide. The oxidation of hydrazine by HDH would make dinitrogen gas and provide the electrons for nitrite

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reduction and hydrazine synthesis (Kartal et al., 2012). Kartal et al. (2011) also proposed that HDH might be the key enzyme responsible for converting N2H4 into the final N2. The total heme c proteins make up 30% of the protein complement of Anammox bacteria, giving their characteristic color that gradually comes to the fore during enrichment (Kartal et al., 2012). It should be noted that the synthesis of heme c requires chelating ferrous iron to form the active regions. However, the Fe(II) concentration chelating with EDTA was almost set as 0.03 mM in almost researches since Van der Graaf et al. (1996) firstly reported the discovery of Anammox process in a fluidized bed reactor. Some researches have reported Fe element could affect metabolism of Anammox bacteria. Van Niftrik et al. (2008) demonstrated that there existed electron-dense particles contained Fe inside Anammoxosome compartment. They proposed that Anammox bacteria stored iron to have an excess supply of Fe for future heme c synthesis. Zhang et al. (2012) demonstrated that Fe-electrode reaction created a favorable environment for the growth of Anammox bacteria. Notably, appropriate increase of Fe(II) from 0.03 to 0.12 mM was proved to be beneficial for more heme c synthesis, hydrazine dehydrogenase (HDH) activity enhancement, and growth rate acceleration for Anammox biomass (Qiao et al., 2013). Thus, it is reasonable to speculate that increase of ferrous iron could promote synthesis of heme c – containing enzymes (such as HDH and HZS), and then accelerate the metabolism of Anammox bacteria, which might promote the growth rate of Anammox biomass. Hitherto, studies conducted on start-up of the Anammox process by increasing Fe(II) concentration in medium have never been reported. Based on these arguments, the main objectives of this study are to: (1) verify the possibility of developing a rapid start-up Anammox reactor via appropriate ferrous iron addition (with a dose of 0.06 and 0.09 mM, respectively) and (2) evaluate the effect of ferrous iron concentration on the quantity of Anammox bacteria, heme c levels, and enzyme activity during the entire operation. 2. Methods 2.1. Anammox start-up seed sludge The anaerobic activated sludge from Lingshui Sewage Treatment Plant (Dalian, China) was inoculated in three upflow column reactors. The seeding sludge had a final mixed liquor suspended solid (MLSS) concentration of about 2.54 g/L in all three reactors. 2.2. Reactors Three identical upflow column reactors R1, R2 and R3 made of polymethyl methacrylate were applied for Anammox start-up, as shown in Fig. 1. The working volumes were 0.5 L with the inner diameter of 5.0 cm and the height of 25.5 cm. The reactor comprised a fluidized bed in the lower part (0–200 mm from the bottom) and a fixed bed in the upper part (200–250 mm from the bottom). The upper part was filled with a non-woven fabric carrier. Six bundles of the carrier were inserted in the upper part with a volume of 0.1 L (20% of the total reactor volume). The cylindrical reactors were equipped with a thermostatic jacket to maintain a fixed temperature of 35 ± 1 °C and covered to protect Anammox bacteria from light. 2.3. Synthetic wastewater The medium used in the experiments mainly consisted of ammonium, nitrite and inorganic carbon source (in the form of (NH4)2SO4, NaNO2 and KHCO3, respectively), as shown in Table 1.

Fig. 1. Schematic diagram of the identical Anammox reactor.

Table 1 Composition of synthesis wastewater. Constituent

Concentration (g/L)

Constituent

Concentration (g/L)

(NH4)2SO4 NaNO2 KHCO3 KH2PO4 KCl

– – 0.125 0.054 0.0014

CaCl2
2H2O NaCl MgSO4
7H2O EDTA
2Na Trace elements solution

0.0014 0.001 0.001 0.005 1.25 ml/L

Table 2 Composition of trace elements solution. Constituent

Concentration (g/L)

Constituent

Concentration (g/L)

EDTA
2Na ZnSO4
7H2O CoCl2
6H2O MnCl2
4H2O CuSO4
5H2O

15 0.43 0.24 0.99 0.25

NaMoO4
2H2O NiCl2
6H2O NaSeO4
10H2O H3BO4 NaWO4
2H2O

0.22 0.19 0.21 0.014 0.05

The composition of the trace mineral medium was as described by Van der Graaf et al. (1996), as depicted in Table 2. The medium for all reactors had the same substrates, only the Fe(II) (sequestrated by EDTA–2Na) concentration were set as 0.03 mM for R1 (control group), 0.06 mM for R2 and 0.09 mM for R3, respectively. The Fe(II) concentration was set based on the previous study (Qiao et al., 2013) in which 0.09 mM of Fe(II) was proved to be the optimistic concentration for Anammox bacteria. The medium was purged with 99.5% N2 to maintain dissolved oxygen below 0.5 mg/L. The pH of the influent was adjusted to 7.0 ± 0.2 by dosing 2M HCl. 2.4. Analytical methods Concentration of nitrite and nitrate were determined by using ion-exchange chromatography (ICS-1100, DIONEX, AR, USA) with an IonPac AS18 anion column after filtration with 0.22 lm pore size membranes. NH4–N and MLSS concentrations were measured according to the Standard Methods (APHA, 1995). pH measurement was done using a digital pH meter (PHS-25, Leici Company,

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China), while DO was measured using a digital DO meter (YSI, Model 55, USA). The heme c content was measured according to the method described in reference (Qiao et al., 2013).

On operational day 1, 50, 100, and 150 d, 2 g (wet weight) biomass was taken from each reactor. The biomass samples were centrifuged at 8000 rpm at 4 °C for 20 min followed by washing twice with sodium phosphate buffer solution (20 mM, pH 7.0). The washed pellets were then resuspended in 20 ml of the same buffer and lysed by freezing and thawing followed by sonication (225 W, at 4 °C for 30 min, Ultrasonic processor CPX 750, USA). Cell mass was separated by centrifugation (22,000 rpm), at 4 °C for 30 min. The supernatant was stored at 20 °C and used as cell extract in the determination of protein and enzyme activity. Protein concentration was measured according to the Bradford procedure (Bradford, 1976), using BSA as a standard. Enzyme activity of hydrazine dehydrogenase (HDH) was determined according to the methods described by Qiao et al. (2013), and the reactions were depicted as an increase in the absorbance of cytochrome c at 550 nm in the standard mixture using a spectrophotometer (V-560 UV/VIS Spectrophotometer, Jasco, Japan). The HDH activity was expressed as lmol of cytochrome c reduced/min/mg protein. 2.6. Quantitative PCR assay Primer pairs Amx 809-F and Amx 1066-R were used for realtime PCR quantification of the Anammox bacteria. The 25 lL reaction volume contained 12.5 lL SYBRÒ Premix Ex Taq™ (TaKaRa, Dalian, China), 0.4 mg/ml bovine serum albumin, 200 nM final concentration of each primer, 0.5 lL Rox reference dye and 2 lL extracted DNA as a template. Three replicates were analyzed for each sample. The PCR program was as follows: denaturation for 2 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, annealing for 30 s at 62 °C and elongation for 30 s at 72 °C. Melting curve analysis showed only one peak at Tm = 87.0 °C. No detectable peaks that were associated with primer-dimer artifacts and no other nonspecific PCR amplification products were observed. The plasmid DNA concentration was determined on a NanodropÒ ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, USA), and the Anammox bacterial 16S rRNA gene copy number was calculated directly from the concentration of extracted plasmid DNA. Sixfold serial dilutions of a known copy number of the plasmid DNA were subjected to q-PCR assay in triplicate to generate an external standard curve. 3. Results 3.1. Reactors performance During inoculation, the reactors were continuously fed with synthetic wastewater to enrich Anammox microorganisms. According to the concentration of effluent ammonium, the startup processes occurred in all three reactors could be divided into phases of cell lysis phase (with effluent ammonium concentration > influent one), lag phase (effluent ammonium concentration stabilized around influent one), activity elevation phase (a rapid decline of effluent ammonium and nitrite concentration simultaneously); the lasting duration of each phase for three reactors are illustrated in Fig. 2. After completing start-up of all three reactors, the operational stability was investigated via increasing the NLR gradually.

Reactor

2.5. Preparation of biomass extracts and determination of enzyme activity

cell lysis phase lag phase activity elevation phase

3

2

1

0

10

20

30

40

50

60

70

Inoculation time (d) Fig. 2. The lasting duration of every phase for each reactor during start-up period. R1, with 0.03 mM Fe(II); R2, with 0.06 mM Fe(II); R3, with 0.09 mM Fe(II).

3.1.1. Start-up period During cell lysis phase, ammonium and nitrite concentration in feeding were keeping around 50 mg/L, and HRT was maintained at 12 h, corresponding to the total NLR about 200 g-N/m3/d. The cell lysis from aerobic bacteria, which cannot adapt to the given conditions, caused conversion from the organic nitrogen to ammonia. Thus, ammonium concentration was even much higher than that of influent when almost all nitrite removal was achieved. Since anaerobic heterotrophic denitrifying bacteria grew much faster than autotrophic Anammox bacteria, denitrifying bacteria might predominate in the first stage. One of the most remarkable features which demonstrated the end of microorganism autolysis was that ammonium concentration in effluent decreased gradually and was close to that in influent at the end of the stage, as depicted in Fig. 3. As shown in Fig. 2, the lasting duration of cell lysis phase for each reactor were 39, 31 and 26 days, respectively. In the lag phase, Anammox activity occurred as both ammonia and nitrite being removed simultaneously. It was also observed that nitrate in effluent increased evidently, indicating the decreasing activity of denitrifying bacteria. Some fluctuations of effluent ammonium concentration were detected, as shown in Fig. 3. However, its average was lower than that of the influent concentration. For R1 (the control, 0.03 mM Fe2+), R2 (0.06 mM Fe2+) and R3 (0.09 mM Fe2+), this phase lasted for 14 days (40–53 d), 13 days (32–44 d) and 8 days (27–34 d), respectively. The average ammonium removal rate for three reactors were calculated as 5.73 ± 2.66, 10.33 ± 2.84 and 16.11 ± 4.98 g-N/m3/d, respectively, suggesting a juvenile Anammox activity in all reactors. The nitrite removal rate of all reactors ranged from 91.57 to 99.65 g-N/m3/d, nevertheless, hardly any nitrate produced. Thus, it was hypothesized that denitrifying bacteria were still the dominant population even though Anammox activity appeared in this phase. The most eminent feature observed in activity elevation phase was the ammonium removal rate increasing rapidly and nitrate accumulating apparently, as shown in Fig. 3. Taking R3 for example, from day 35 to 50 (16 days), the ammonium removal efficiency shot up from 24.46 to 80.92 g-N/m3/d, while the nitrite removal rate reached 81.56 g-N/m3/d. From day 50, both ammonium and nitrite concentration in effluent of R3 were stable lower than 10 mg/L. At the end of this phase, the stoichiometric molar ratio of nitrite consumption and nitrate production versus ammonium consumption were calculated as 1.25 ± 0.10 and 0.26 ± 0.07, respectively. It is indicated that Anammox process was successfully started up from conventional activity sludge in R3. Similarly, the activity elevation phase for R1 and R2 lasted for 17 days (54– 70 d) and 15 days (45–59 d), respectively, shown in Fig. 2. The stoichiometric molar ratio of nitrite consumption and nitrate production to ammonium consumption were 1.05 ± 0.15 and 0.20 ± 0.02 for R1; 1.16 ± 0.20 and 0.19 ± 0.06 for R2, respectively.

N concentration(mg/L)

Z. Bi et al. / Bioresource Technology 170 (2014) 506–512

in R1 and R2. For R1, the NO2 –N in effluent increased to around 40 mg/L when the NLR was about 1100 g-N/m3/d. Similarly, NO2 –N in effluent of R2 was even higher than 50 mg/L when NLR reached 1400 g-N/m3/d. In contrast, the effluent NO2 –N concentration of R3 was almost kept below 20 mg/L during the same high NLRs, shown in Fig. 4(b). This phenomenon could be attributed to the relatively higher Anammox bacteria activity and stronger stability against the high NLR impacting in R3, which was closely related to the higher influent Fe(II) concentration.

R1

120

509

90 60 30 0

N concentration(mg/L)

3.2. Real-time PCR experimental results In order to clarify the effects of Fe(II) on the growth rates of Anammox bacteria, the real-time PCR (qPCR) experiments were conducted on the operational day 1, 50, 100 and 150 as shown in Fig. 5. The copies number of Anammox bacteria 16S rRNA gene in all reactors increased with the enrichment process, which was consistent with the nitrogen removal performance and Anammox activity during the entire experiments. However, the copies number of R3 was considerably higher than that of other two reactors. On day 150, the copies number in R3 was 1.97  109 copies/g biomass, around 1.73- and 1.32-fold as much as those in R1 and R2, respectively. These results demonstrated that the quantity of Anammox bacteria in R3 was much larger than those of R1 and R2, which further promoted the fast start-up of R3.

R2

120 90 60 30

N concentration(mg/L)

0

R3

120 90

3.3. Heme c content and crude enzymes (HDH) activities 60 30 0 0

15

30

45

60

75

Inoculation time(d) +

NO 2-N (inf)

+

NO 2-N (eff)

NH 4 -N (inf) NH 4 -N (eff)

NO 3-N (eff)

Fig. 3. Time course of influent and effluent N concentration of each reactor during start-up period. R1, with 0.03 mM Fe(II); R2, with 0.06 mM Fe(II); R3, with 0.09 mM Fe(II).

The entire start-up time was shorten to 50 d in R3 (with 0.09 mM Fe2+) and 59 d in R2 (with 0.06 mM Fe2+) compared with 70 d in R1 (with 0.03 mM Fe2+). Notably, the significant reduction of start-up time occurred in cell lysis phase and lag phase, while the activity elevation phase almost lasted for the same period, as shown in Fig. 2. 3.1.2. Operational stability investigation From day 71–150, influent ammonium and nitrite concentration increased stepwise from 50 to 170 mg/L and HRT was maintained at 6.67 h, corresponding to the total nitrogen loading rate (NLR) were ranging from 365 to 1400 g-N/m3/d for three reactors. In the following operation, the NRP of R3 was more stable and efficient compared to the other two reactors. On day 150, the average NRR of R3 and R2 were calculated as 1193 and 929 g-N/m3/d, while the average NRR of R1 was 713 g-N/m3/d, as shown in Fig. 4(a). These values of R3 and R2 were about 77.2% and 43.22% higher than that of R1. During this period, we tried to depress the effluent NO2 –N below 20 mg/L in order to eliminate or mitigate the maleffects of NO2 –N on Anammox biomass. Thus, the maximum influent ammonium/nitrite concentration for R3 and R2 reached 170/ 220 mg/L, while only 130/170 mg/L for R1. However, the relatively higher NLRs still caused the higher effluent NO2 –N concentrations

The heme c contents of three reactors were measured on day 1, 50, 100 and 150 as shown in Fig. 6(a). The changes of heme c level in each reactor were also consistent with NRP. During the whole operational period, samples from R3 exhibited the highest heme c content. At the end of experiment, the heme c level of R3 reached 0.14 lmol heme c/mg protein, which was 1.47 and 1.31 folds as much as those of R1 and R2, respectively. According to Fig. 6(a), it could be speculated that the more increase of heme c contents in R2 and R3 were closely associated with the relatively higher influent Fe(II) concentration. Anammox bacteria rely on a specific set of cytochrome c-type proteins, in particular hydroxylamine oxidoreductase-like octaheme proteins (HAOs), hydrazine synthase (HZS) for their metabolism. In Anammox process, both hydrazine and NO were considered the intermediates when Anammox bacteria converted ammonium to dinitrogen gas. The oxidation of hydrazine by an HAO-like protein called hydrazine dehydrogenase (HDH) would make dinitrogen gas and provide the electrons for nitrite reduction and hydrazine synthesis (Kartal et al., 2012). Kartal et al. (2011) also proposed that HDH might be the key enzyme responsible for converting N2H4 into the final N2. In this study, the crude enzyme (HDH) activities of three reactors were also investigated on day 1, 50, 100 and 150 to clarify the effects of Fe(II) on Anammox activity, illustrated in Fig. 6(b). Samples from R3 exhibited relatively higher HDH activity all the time, which was closely consistent with the NRP. On day 150, HDH activity of R3 was 3.67 ± 0.27 lmol cytochrome c reduced/ min/mg, which was approximately 1.42- and 1.20-fold as much as those of R1 and R2, respectively. It is proposed that the relatively higher HDH activity could be one of the major reasons that R3 was more stable and effective during the whole operation. 4. Discussion The observed three phases of Anammox start-up process in this study were also reported by other researchers (Wang et al., 2012; Tang et al., 2009). The Anammox process appeared on day 26 and

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(a)

(b) R1

N concentration(mg/L)

3

NLR/NRR(g-N/m /d)

1600 1200 800 400

N concentration(mg/L)

3

NLR/NRR(g-N/m /d)

R2

1200 800 400 0 100

125

120

60

240

R2

180

120

60

150

R3

N concentration(mg/L)

3

NLR/NRR(g-N/m /d)

180

0

75 1600

R1

0

0 1600

240

1200 800 400

240

R3

180

120

60

0

0 75

100

125

150

75

100

Inoculation time(d) +

NO2-N (inf)

+

NO2-N (eff)

NH4 -N (inf)

NLR (nitrogen loading rate)

125

150

Inoculation time(d)

NRR (nitrogen removal rate)

NH4 -N (eff)

NO3-N (eff)

Fig. 4. Profile of N removal of each reactor during day 71–150 (a) time courses of NLR and NRR performance; (b) time course of influent and effluent N concentration. R1, with 0.03 mM Fe(II); R2, with 0.06 mM Fe(II); R3, with 0.09 mM Fe(II).

Gene copies (copies/g)

3.0x10

9

2.5x10

9

2.0x10

9

1.5x10

9

1.0x10

9

5.0x10

8

R1 R2 R3

0.0 1

50

100

150

Inoculation time(d) Fig. 5. Comparison of Anammox bacteria 16S rRNA gene copies number of each reactor. Each measurement was carried out in triplicate. R1, with 0.03 mM Fe(II); R2, with 0.06 mM Fe(II); R3, with 0.09 mM Fe(II).

was successfully started up within 50 days in R3, which was considered to be much shorter than that in other literatures, for example 120–200 d of UASB (Yang et al., 2009), 100–120 d of SBR (Third et al., 2005), 78 d of granular SBR (Lopez et al., 2008). The remarkable reduction of Anammox start-up period occurred in cell lysis

phase and lag phase, which may be closely associated with the relatively higher Fe(II) concentration in media. Iron is virtually an important micronutrient for all living organisms except lactic acid bacteria, where manganese and cobalt are used in place of iron. It is well established that bacterial growth and subsequent colonization is dependent on the ability to acquire iron. Under physiological conditions, iron can exist in either the reduced ferrous (Fe2+) form or the oxidized ferric (Fe3+) form. The redox potential of Fe2+/Fe3+ makes iron extremely versatile, when it is incorporated into proteins as a catalytic center or as an electron carrier (Krewulak and Vogel, 2008). Thus iron is important for numerous biological processes which include photosynthesis, respiration, the tricarboxylic acid cycle, oxygen transport, gene regulation, DNA biosynthesis, etc. Insertion of a ferrous iron atom into the porphyrin macrocycle by the enzyme ferrochelatase creates heme, which is a common cofactor used for a multitude of biological reactions. For Anammox bacteria, heme can provide catalytic and electron transfer operations, also serve a critical role in the assembly of major enzyme complexes, including HDH, HZS, NirS (nitrite reductase), HAO, cytochrome bc1 complex, etc. In the absence of heme, the assembly of such enzymes would be impaired. On the basis of experimental data, apparently, the amount of available ferrous iron in medium could affect these processes directly. Since Anammox process was discovered in 1996, the Fe(II) (chelating with EDTA) concentration in culture medium was almost set as 0.03 mM, which was probably insufficient for Anammox biomass, hampering sufficient

Z. Bi et al. / Bioresource Technology 170 (2014) 506–512

5. Conclusions

(a) 0.25

The start-up time of Anammox reactor from conventional activity sludge could be reduced significantly with elevating Fe(II) concentration. As Fe(II) concentration was ranged 0.03–0.09 mM, the growth rate of Anammox bacteria in R3 (with 0.09 mM Fe2+) and R2 (with 0.06 mM Fe2+) was much faster than the control group (with normal 0.03 mM Fe2+). It was confirmed that both heme c synthesis and HDH activity were highly corresponded to increase of external Fe(II) dosage, which was regarded as the intrinsic factor for accelerating the start-up period of Anammox process.

R1 R2 R3

Heme-C concentration µmol/mg

0.20

0.15

0.10

Acknowledgements 0.05

0.00

1

100

50

150

Inoculation time(d)

(b) 5

4

3

2

1

0

1

This work was supported by the Natural Science Foundation of China (Nos. 21377014, 51008045), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. QAK201305), Dalian Municipal Science and Technology Projects (No. 2012E11SF067) and Fundamental Research Funds for the Central Universities (No. DUT12LK20). References

R1 R2 R3

Crude enzyme activities (µmol cyto c reduced/min/mg)

511

100

50

150

Time(d) Fig. 6. Comparison of (a) heme c contents and (b) HDH activities of each reactor. Each measurement was carried out in triplicate. R1, with 0.03 mM Fe(II); R2, with 0.06 mM Fe(II); R3, with 0.09 mM Fe(II).

heme c synthesis. Moreover, heme can be utilized as an iron reserve for prokaryota, which was also correlated with experimental results of this study. The intracellular heme concentration of Anammox biomass exhibited an elevatory trend following with the increasing ferrous iron in medium. Hence, the proposed scenario of this research is that the functional importance of iron, coupled with its generally poor bioavailability, makes it essential for Anammox bacteria to husband this element. Consequently, adequate ferrous iron can support production of heme c, which further guarantee synthesis of sufficient heme-proteins and make them become more active. Ultimately, the growth rate of Anammox bacteria was accelerated and quick start-up of Anammox reactor was achieved. In this study, we supported the point that different external Fe(II) concentrations could cause discrepant increase of heme c synthesis, and significantly accelerate the enrichment of Anammox biomass from activity sludge. Therefore, increased Fe(II) dosage in feeding could be the intrinsic causes for favoring the Anammox bacteria enrichment. Considering the long period for Anammox reactor starting-up, our findings should be of great help to gain new and promising insight into the ferrous iron effects on acceleration of Anammox bacteria enrichment.

APHA, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association, New York. Bradford, M.M., 1976. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chen, C.J., Huang, X.X., Lei, C.X., Zhu, W.J., Chen, Y.X., Wu, W.X., 2012. Improving Anammox start-up with bamboo charcoal. Chemosphere. 89, 1224–1229. Jetten, M.S.M., Wagner, M., Fuerst, J., Loosdrecht, M.V., Kuenen, J.G., Strous, M., 2001. Microbiology and application of the anaerobic ammonium oxidation (Anammox) process. Curr. Opin. Biotechnol. 12, 283–288. Jin, R.C., Zheng, P., Hu, A.H., Mahmood, Q., Hu, B.L., Jilani, G., 2008. Performance comparison of two Anammox reactors: SBR and UBF. Chem. Eng. J. 138, 224– 230. Kartal, B., Maalcke, W.J., de Almeida, N.M., Cirpus, I., Gloerich, J., Geerts, W., den Camp, H.J.M. Op, Harhangi, H.R., Janssen-Megens, E.M., Francoijs, K.J., Stunnenberg, H.G., Keltjens, J.T., Jetten, M.S.M., Strous, M., 2011. Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130. Kartal, B., van Niftrik, L., Keltjens, J.T., den Camp, H.J. Op, Jetten, M.S., 2012. Anammox-growth physiology, cell biology, and metabolism. Adv. Microb. Physiol. 60, 211–262. Kieling, D.D., Reginatto, V., Schmidell, W., Travers, D., Menes, R.J., Soares, H.M., 2007. Sludge washout as strategy for Anammox process start-up. Process Biochem. 42, 1579–1585. Krewulak, K.D., Vogel, H.J., 2008. Structural biology of bacterial iron uptake. Biochim Biophys Acta. 1778, 1781–1804. Kuenen, J.G., 2008. Anammox bacteria: from discovery to application. Nat. Rev. Microbiol. 6, 320–326. Lopez, H., Puig, S., Ganigue, R., Ruscalleda, M., Balaguer, M.D., Colprim, J., 2008. Start-up and enrichment of a granular Anammox SBR to treat high nitrogen load wastewaters. J. Chem. Technol. Biotechnol. 83 (3), 233–241. Qiao, S., Bi, Z., Zhou, J.T., Cheng, Y.J., Zhang, J., 2013. Long term effects of divalent ferrous ion on the activity of anammox biomass. Bioresour. Technol. 142, 490– 497. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589– 596. Tang, C.J., Zheng, P., Mahmood, Q., Chen, J.W., 2009. Start-up and inhibition analysis of the Anammox process seeded with anaerobic granular sludge. J. Ind. Microbiol. Biotechnol. 36, 1093–1100. Tao, Y., Gao, D.W., Fu, Y., Wu, W.M., Ren, N.Q., 2012. Impact of reactor configuration on Anammox process start-up: MBR versus SBR. Bioresour. Technol. 104, 73–80. Third, K.A., Paxman, J., Schmid, M., Strous, M., Jetten, M.S.M., Cord-Ruwisch, R., 2005. Enrichment of Anammox from activated sludge and its application in the CANON process. Microb. Ecol. 49 (2), 236–244. Van der Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142, 2187–2196. Van der Star, W.R.L., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi, T., Strous, M., Picioreanu, C., van Loosdrecht, M.C.M., 2007. Startup of reactors for anoxic ammonium oxidation: experiences from the first full-scale anammox reactor in Rotterdam. Water Res. 41, 4149–4163. van Loosdrecht, M.C.M., 2008. Innovative nitrogen removal. In: Henze, M., van Loosdrecht, M.C.M., Ekama, G.A., Brdjanovic, D. (Eds.), Biological Wastewater Treatment: Principles, Modeling and Design. IWA Publishing, London, pp. 139– 154.

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Z. Bi et al. / Bioresource Technology 170 (2014) 506–512

Van Niftrik, L., Geerts, W.J.C., van Donselaar, E.G., Humbel, B.M., Yakushevska, A., Verkleij, A.J., Jetten, M.S.M., Strous, M., 2008a. Combined structural and chemical analysis of the anammoxosome: a membrane-bounded intracytoplasmic compartment in anammox bacteria. J. Struct. Biol. 161, 401– 410. Wang, T., Zhang, H., Gao, D., Yang, F., Zhang, G., 2012. Comparison between MBR and SBR on Anammox start-up process from the conventional activated sludge. Bioresour. Technol. 122, 78–82.

Wang, T., Zhang, H.M., Yang, F.L., Li, Y., Zhang, G., 2013. Start-up and long-term operation of the Anammox process in a fixed bed reactor (FBR) filled with novel non-woven ring carriers. Chemosphere. 91, 669–675. Yang, Z.Q., Zhou, S.Q., Sun, Y.B., 2009. Start-up of simultaneous removal of ammonium and sulfate from an anaerobic ammonium oxidation (Anammox) process in an anaerobic up-flow bioreactor. J. Hazard. Mater. 169 (1), 113–118. Zhang, J.X., Zhang, Y.B., Li, Yang., Zhang, L., Qiao, S., Yang, F.L., Quan, X., 2012. Enhancement of nitrogen removal in a novel anammox reactor packed with Fe electrode. Bioresour. Technol. 114, 102–108.