N2O emission from a partial nitrification–anammox process and identification of a key biological process of N2O emission from anammox granules

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 4 6 1 e6 4 7 0

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

N2O emission from a partial nitrificationeanammox process and identification of a key biological process of N2O emission from anammox granules Satoshi Okabe a,b,*, Mamoru Oshiki a,b, Yoshitaka Takahashi a, Hisashi Satoh a a

Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan b Japan Science and Technology Agency, CREST, Japan

article info

abstract

Article history:

Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern.

Received 11 April 2011

The emission of N2O from a lab-scale two-reactor partial nitrification (PN)eanammox reactor

Received in revised form

was therefore determined in this study. The average emission of N2O from the PN and

25 June 2011

anammox process was 4.0
1.5% (9.6
3.2% of the removed nitrogen) and 0.1
0.07%

Accepted 20 September 2011

(0.14
0.09% of the removed nitrogen) of the incoming nitrogen load, respectively. Thus,

Available online 29 September 2011

a larger part (97.5%) of N2O was emitted from the PN reactor. The total amount of N2O emission from the PN reactor was correlated to nitrite (NO 2 ) concentration in the PN effluent rather

Keywords:

than DO concentration. In addition, further studies were performed to indentify a key bio-

Partial nitrification

logical process that is responsible for N2O emission from the anammox process (i.e., granules).

Anammox

In order to characterize N2O emission from the anammox granules, the in situ N2O production

Nitrous oxide emission

rate was determined by using microelectrodes for the first time, which was related to the

Granules

spatial organization of microbial community of the granule as determined by fluorescence in

Microelectrodes

situ hybridization (FISH). Microelectrode measurement revealed that the active N2O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N2O production zone. Anammox bacteria were present throughout the granule, whereas ammonium-oxidizing bacteria (AOB) were restricted to only the granule surface. In addition, addition of penicillin G that inhibits most of the heterotrophic denitrifiers and AOB completely inhibited N2O production in batch experiments. Based on these results obtained, denitrification by putative heterotrophic denitrifiers present in the inner part of the granule was considered the most probable cause of N2O emission from the anammox reactor (i.e., granules). ª 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Nitrous oxide (N2O) has a more than 300-fold greater potential for global warming effects than carbon dioxide, even though N2O only accounts for approximately 0.03% of total

greenhouse gas emissions (Bates et al., 2008). Thus, the actual impact of N2O on global warming has been estimated up to 10% of total greenhouse gas emissions. It also takes part in stratospheric ozone depletion and is toxic to humans. Wastewater treatment systems, especially, biological nitrogen

* Corresponding author. Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan. Tel./fax: þ81 (0)11 706 6266. E-mail address: [email protected] (S. Okabe). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.09.040

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removal processes, have been known to be a potential N2O emission source. It is, therefore, in urgent need of reducing the emission and of identifying the factors that control the emission of N2O from wastewater treatment plants (WWTPs). Several measurements at lab-scale and full-scale WWTPs have indicated that N2O can be produced in substantial amounts from biological nitrogen removal processes (Foley et al., 2010; Osada et al., 1995; Tallec et al., 2006; Kampschreur et al., 2008, 2009b). Both nitrification and denitrification processes can lead to emission of N2O. However, N2O emissions are extremely variable and depend on many operational parameters such as dissolved oxygen (DO) and nitrite (NO 2 ) concentrations in both nitrification and denitrification stage (Beline et al., 2001; Gejlsbjerg et al., 1998; Itokawa et al., 2001; Kampschreur et al., 2008; Park et al., 2000) and carbon availability (low chemical oxygen demand (COD)/N ratio) in the denitrification stage (Itokawa et al., 2001; Park et al., 2000). A recent review by Kampschreur et al. (2009a) showed that there are large variations in the N2O emissions from full-scale WWTPs (0e14.6% of the nitrogen load) and lab-scale WWTPs (0e95% of the nitrogen load). Recently, sustainable wastewater treatment systems that can minimize energy consumption, emission of greenhouse gases, and sludge production have been attracting the attention. A nitrogen removal process via anaerobic ammonium oxidation (anammox) has been recognized as a promising costeffective and low energy alternative to the conventional nitrificationedenitrification processes due to a significant reduction of aeration and external carbon source (van Dongen et al., 2001; Kartal et al., 2010). In nitrogen removal via anammox process, ammonium in wastewater is partly pre-oxidized to nitrite (i.e., partial nitrification) by ammonium-oxidizing bacteria (AOB) before feeding into the anammox process. The produced nitrite together with remaining ammonium is then converted to dinitrogen gas (N2) in the anammox process. In the two-reactor partial nitrificationeanammox process, significant N2O production could be expected during the partial nitrification due to accumulation of high NO 2 and DO-limited conditions. In addition, N2O emission can also be expected from the anammox process since the anammox processes have been generally operated at high volumetric nitrogen removal load as described by Tsushima et al. (2007) and Tang et al. (2011) and at low COD/N ratio, even though anammox bacteria have not been shown to produce N2O under physiological conditions. Emission of N2O from a full-scale tworeactor partial nitrificationeanammox process treating reject water was determined to be 2.3% of the total nitrogen load (1.7% in the partial nitrification process and 0.6% in the anammox process) (Kampschreur et al., 2008). Emission of N2O from a full-scale single-stage partial nitrificationeanammox reactor treating wastewater from a potato processing factory and reject water of a municipal sludge dewatering plant was 1.2% of the total nitrogen load (Kampschreur et al., 2009b), which is higher than the emission from a lab-scale single reactor partial nitrificationeanammox system on artificial wastewater (less than 0.1% of the nitrogen load) (Sliekers et al., 2002). The magnitude and source of N2O emission in the combined partial nitrification and anammox process are, however, relatively unknown, especially, the potential and

mechanism of N2O emission from anammox reactors or granules (or biofilms) is also unknown. Emission of N2O from an energy-saving and cost-effective partial nitrificationeanammox process would hamper the practical application and should therefore be avoided. In this study, a lab-scale partial nitrificationeanammox process was developed in two separate reactors to investigate N2O emission from both processes. In addition, further studies were performed to indentify a key biological process that is responsible for N2O emission from the anammox process (i.e., granules). In order to characterize N2O emission from the anammox granules, microelectrodes were used to determine in situ N2O production rate, which was related to spatial organization of microbial community of the granule analyzed by fluorescence in situ hybridization (FISH).

2.

Materials and methods

2.1.

Lab-scale partial nitrification reactor

An up-flow biofilm partial nitrification (PN) reactor with a working volume of 800 cm3 and nonwoven fabric sheets (4.0  4.0  0.8 cm  18 sheets; Japan Vilene Co., Ltd., Tokyo, Japan) as support material for biofilms was used. The PN reactor was established and operated for 680 days as described previously (Cho et al., 2011; Okabe et al., 2011). Synthetic nutrient medium (Okabe et al., 2011) and air was supplied continuously from the bottom of the reactor. Although the dissolved oxygen concentration (DO) was not controlled during the experiment, the air-flow rate was adjusted in the range 100e650 mL min1. The incubation temperature was maintained at 35  C. The influent pH was adjusted to 7.8
0.1. The hydraulic retention time (HRT) of the reactor was fixed at 4 h.

2.2.

Anammox reactor

An up-flow granular-sludge anammox reactor with a working volume of 150 cm3 has been stably operated at 35  C for more than 2 years (Cho et al., 2010). This reactor was originally inoculated with anammox biomass taken from an anammox reactor (a maximum nitrogen removal rate of 34.2 kgN m3 d1) developed previously in our laboratory (Kindaichi et al., 2007; Tsushima et al., 2007). Only the reactor performance after about 2 years is presented.

2.3.

Partial nitrificationeanammox process

After achieving stable partial nitrification (after 680 days) and anammox reaction (after approximately 2 years), the PN reactor was combined with the anammox reactor. The half amount of ammonium in the influent was oxidized to nitrite in the PN reactor, resulting in the ammonium and nitrite ratio of about 1:1 in the effluent. The effluent of the PN reactor was introduced into the anammox reactor via a flow equalizing tank (500 mL), in which dissolved oxygen (DO) carried over from the partial nitrification reactor are removed, and pH was adjusted to around 7.2. Flow rate into the anammox reactor was set to obtain a HRT 0.3e0.8 h. After stable performance of the combined PN and anammox reactor was achieved, N2O

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 4 6 1 e6 4 7 0

was measured in the off-gas and liquid phase of the PN and anammox reactor. The off-gas stream from the anammox reactor was generated by the gas production.

2.4.

Analytical procedure

To monitor the performance of partial nitrificationeanammox reactor, one grab influent and effluent sample was collected at regular time interval during the operation. Ammonium (NHþ 4 N), nitrite (NO 2 -N), and nitrate (NO3 -N) in the influent and effluent were measured three times by using ion-exchange chromatography (DX-100, DIONEX, CA., USA) with an IonPac CS3 cation column and IonPac AS9 anion column after filtration with 0.2-mm pore size membranes (ADVANTEC, Tokyo, Japan). Analytical errors were within 5% for each chemical during the experiment. Dissolved oxygen (DO) concentration in the effluent was measured by using a DO meter (DO-5Z, KRK, Japan).

2.5.

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anammox granules, penicillin G (500 mg L1) was added directly to the medium to inhibit the activity of the peptidoglycan-containing bacteria, but not anammox bacteria (van de Graaf, et al., 1996). The bottles were then incubated at 35  C. Gas and medium samples were taken for chemical analyses at appropriate time intervals.

2.7.

Fixation and cryosectioning of biofilm samples

Granule samples obtained from the anammox reactor were fixed in a 4% paraformaldehyde solution for 24 h at 4  C, washed three times with phosphate-buffer saline (PBS) (10 mM sodium phosphate buffer, 130 mM sodium chloride; pH 7.2), and embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) overnight to infiltrate the OCT compound into the biofilm, as described previously (Okabe et al., 1999a). After rapid freezing at 21  C, 10- to 20-mm-thick vertical thin sections were prepared with a cryostat (Reichert-Jung Cryocut 1800, Leica, Bensheim, Germany) (Okabe et al., 1999b)

N2O measurement 2.8.

The off-gas grab samples were collected from the PN and anammox reactor with a gas-tight syringe. The N2O concentration in the off-gas was measured with a GC-12A gas chromatograph (Shimadzu, Japan) equipped with an electron capture detector (ECD) and using nitrogen gas as carrier gas. Temperatures of the injector, column, and detector were 330, 60, and 330  C, respectively. The dissolved N2O gas concentration in the liquid phase was measured by using the headspace method. Briefly, a sample was transferred to a 70-mL glass vial. The vial was sealed by a butyl-rubber stopper and aluminum cap. After the glass vial was shaken for a few minutes, N2O in the gas phase was measured by the gas chromatograph as described above. The N2O dissolved in the liquid phase was calculated by the solubility formula of Weiss and Price (1980). For calculation of N2O emission rate from the process, the N2O emission rate (mg-N m3 d1) was calculated relative to the nitrogen load into the partial nitrification reactor and the nitrogen conversion rate of each reactor, respectively.

2.6. Batch experiments for estimating N2O emission characteristics

The 16S rRNA-targeted oligonucleotide probes used in this study were follows; EUB mix probe (EUB338, EUB338II, and EUB338III) for all bacteria (Daims et al., 1999), which were used in an equimolar, Amx820 for Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (Schmid et al., 2001), Nse1472 for Nitrosomonas europaea, Nitrosomonas halophila, and Nitrosomonas eutropha, Nsv443 for Nitrosospira spp. (Mobarry et al., 1996) and Nso190 for ammonia-oxidizing bproteobacteria (Mobarry et al., 1996). The probes were labeled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine 5-isothiocyanate (TRITC) at the 50 end. In situ hybridization was performed according to the procedure described by Okabe et al. (1999b). A model LSM510 confocal laser-scanning microscope (CLSM, Carl Zeiss, Oberkochen, Germany), equipped with an Ar ion laser (488 nm) and HeNe laser (543 nm), was used. The average surface area fraction of probe-hybridized cells was determined from at least 10 representative LSM projection images of each cross-section of the biofilm samples using image analysis software provided by Zeiss (Okabe et al., 2004).

2.9. For batch experiments, anammox biomass taken from the anammox reactor was disrupted by intensive magnetic stirring to reduce mass transfer limitation at low substrate concentrations. For each batch experiment, 800 mL of disrupted anammox biomass (the aggregate diameter <100 mm) was mixed with 20 mL of the anammox nutrient medium in 34 mL serum bottles (ca. 0.2 g-VSS L1). The serum bottles were sealed with butyl-rubber stoppers and purged with N2 gas (99.99%) to remove oxygen. The anammox nutrient medium consisted of 120 mg-N L1(NH4)2SO4, 100 mg-N L1 NaNO2, 500 mg L1 KHCO3, 27 mg L1 KH2PO4, 300 mg L1 MgSO4$2H2O, 180 mg L1 CaCl2$2H2O, and 1 mL of trace element solution I and II (van de Graaf et al., 1996). The same medium was used for all batch experiments. pH of the medium was adjusted at 7.2. To identify the key biological process that is responsible for N2O production in the

Fluorescence in situ hybridization (FISH)

Microelectrode measurements

N2O and NHþ 4 concentration profiles in anammox granules were measured using Clark-type microelectrodes and LIXtype microelectrodes, respectively. N2O microelectrode was purchased from Unisense (Arhus, Denmark) and calibrated according to the instruction provided by Unisense. The LIX microelectrodes were prepared, calibrated, and operated as described by Okabe et al. (1999a) and Satoh et al. (2003). For microelectrode measurements, the granules with diameters of 2e3 mm were selected and positioned, using five insect needles, in the flow chamber (4.0 L) that was filled with the synthetic medium at 35  C (Satoh et al., 2007). The anammox granules used for microelectrode measurements were bigger than the mode of the granule size distribution (1.5e2.0 mm). The medium used for microelectrode measurement was the same as the synthetic nutrient medium fed to the PN reactor

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 except for NHþ 4 and NO2 concentrations, which were both 4.5 mM. The medium in the flow chamber was kept anaerobic by continuous bubbling with N2 gas (99.9%), which also provided sufficient mixing of the medium. The granules were acclimated in the medium at least 3 h to ensure that steady-state profiles were obtained. At least three profiles were measured for each chemical. A concentration profile was measured only once in a granule due to deterioration of anammox activity during the measurement. The net specific consumption or production rates of NHþ 4 and N2O were calculated from the mean concentration profiles by using Fick’s second law of diffusion as described by Kindaichi et al. (2007). Molecular diffusion coefficients of 5 cm2 s1 for N2O in 1.38  105 cm2 s1 for NHþ 4 and 2.10  10  water at 35 C were used for the calculation (Laverman et al., 2007).

3.

ALR was decreased to about 2.5 kg-N m3 d1 by reducing the influent flow rate (corresponding to HRT of 3.4 h) in order to þ achieve the stable effluent NO 2 -N/NH4 -N ratio of 1:1. The airflow rate was also reduced accordingly. Although the operational parameters were changed, the effluent concentrations of   NHþ 4 , NO2 , and NO3 were relatively stable during N2O  measurement (Fig. 1A). The effluent NHþ 4 and NO2 concentra1 tions were in the range of 100e222 mg-N L and 80e180 mgN L1, respectively. Approximately 40% of influent ammonium was oxidized to nitrite, resulting in an average nitrite production rate (NIPR) of 1.15
0.35 kg-N m3 d1 (Fig. 1B). The NIPR of the PN reactor was limited by air-flow rate in this study (Fig. S1). The nitrate production rate (NAPR) was negligible (0.02
0.01 kg-N m3 d1) during this period. Dissolved oxygen (DO) concentration fluctuated but was below 2.0 mg O2 L1 after the air-flow rate was reduced to around 200 mL h1 (day 33) (Fig. 1C). During the measurement period, 100% of the effluent of the PN reactor was continuously fed into the anammox reactor. The performance of the anammox reactor is shown in Fig. 2. The anammox reactor has been operated more than 2 years in advance and only the reactor performance was shown during N2O measurement. The anammox reactor was operated at high nitrogen loading rate (NLR) of 21.5
2.0 kg-N m3 d1 for initial 40 days, and the NLR was decreased to 11.6
1.2 kgN m3 d1 by reducing the influent flow rate to the half (Fig. 2C). The nitrogen removal rate (NRR) fluctuated (7.5e15.0 kgN m3 d1) during the initial 40 days, but thereafter it became stable (6.9
1.2 kg-N m3 d1).

Results and discussion

3.1. Performance of the partial nitrification (PN) and anammox reactor The partial nitrification (PN) reactor has been operated more than 680 days in advance and only the PN reactor performance during N2O measurement (after 680 days) is shown in Fig. 1. The PN reactor was operated at a high ammonium loading rate (ALR) of 3.5e4.1 kg-N m3 d1 (a constant influent ammonium concentration of 300 mg-N L1). Thereafter (after 33 days), the

A

B

C

D

D L Fig. 1 e The performance of the partial nitrification reactor: (A) concentrations of NHD 4 -N in influent, and NH4 -N, NO2 -N, and L NO3 -N in effluent, (B) ammonium loading rate (ALR), nitrite production rate (NIPR), nitrate production rate (NAPR), and N2O emission rate (kg mL3 dayL1) (C) influent flow rate, air-flow rate, and dissolved oxygen (DO) concentration, and (D) N2O concentrations in the off-gas and in the effluent of the partial nitrification reactor.

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NH4+

NO2-

B

NO3-

200 150 100 50

250 NH4+

100 50 0

Time (day)

Time (day)

25.0 20.0

300 15.0 200 10.0

N2O in liquid (µM)

D

30.0 Inf luent f low rate NLR NRR

NRR (kg-TN m-3 day -1)

Inf luent f low (mL h-1)

500 400

NO3-

150

0

C

NO2-

200

200

1600 N2O liquid

1200

100

800

1000

600 400

5.0

0

0.0

Time (day)

1400

150

50

100

N2O gas

N2O in of f gas (ppm)

250

Concentrations (mg-N L-1)

Concentrations (mg-N L-1)

A

200 0

0

Time (day)

L L Fig. 2 e The performance of the granular-sludge anammox reactor: (A) concentrations of NHD 4 -N, NO2 -N, and NO3 -N in D L L influent, (B) concentrations of NH4 -N, NO2 -N, and NO3 -N in effluent, (C) influent flow rate, nitrogen loading rate (NLR), and nitrogen removal rate (NRR), and (D) N2O concentrations in the off-gas and in the effluent of the anammox reactor.

3.2. N2O emission from the partial nitrificationeanammox process N2O concentrations in the off-gas and effluent of both reactors were measured, respectively (Figs. 1D and 2D). The N2O concentration in the off-gas of the PN reactor varied widely (79e645 ppm) probably due to the fluctuation of the air-flow rate, while the N2O concentration in the liquid phase was around 1.0 mM during the measurement (Fig. 1D). This result indicates that most of the produced N2O was stripped out from liquid phase by aeration. Thus, total amount of N2O emission from the PN reactor was determined by multiplying N2O concentration in the off-gas by air-flow rate. Since the ammonium loading rate and air-flow rate of the PN reactor were changed at 33 days, only the N2O emission data obtained after 33 days were used for the calculation. The total amount of N2O emission from the PN reactor seemed to respond to the 2 NO 2 concentration (r ¼ 0.61 (P < 0.05)) (Fig. S2A) in the effluent of the PN reactor, which corresponds to the previous findings by Kampschreur et al. (2008, 2009a). However, it should be noted that since only grab samples were taken and measured at given time intervals in this study, further studies will be needed to obtain clear correlations. On the other hand, there was no significant correlation between the N2O levels in the off-gas and DO concentration (r2 ¼ 0.18 (P > 0.5)) (Fig. S2B). Since the PN reactor was made up of relatively thick biofilms, anoxic zones could be developed in the biofilms regardless of the fluctuation of DO concentrations, leading to N2O production in the anoxic zones. Thus, it is thought that there was no

clear relation between the total amount of N2O emission and DO concentrations in this study. In the anammox reactor, the N2O concentration in the offgas fluctuated widely (93e1358 ppm) probably due to the changes in influent flow rate and NLR (Fig. 2D). The N2O levels in liquid and gas phases after 33 days were taken into account for nitrogen balance of the overall PNeanammox system (Fig. 3). In the PN reactor, on average 4.0
1.5% of the incoming nitrogen load (or 9.6
3.2% of the removed nitrogen in the PN reactor) was converted to N2O. On the other hand, the average emission of N2O from the anammox reactor was 0.1
0.07% of the incoming nitrogen load (0.14
0.09% of the removed nitrogen in the anammox reactor). Thus, the larger part (97.5%) of N2O was emitted from the PN reactor (Fig. 3). Based on a nitrogen mass balance, about 75% of the nitrogen load was removed from the water phase as N2 gas in this system (N2 gas was not measured in this study); the remaining 25% was present in the effluent as   NHþ 4 (10%), NO2 (6%), and NO3 (9%). The N2O emission level from the lab-scale PN process (4.0
1.5% of the incoming total nitrogen load) and anammox process (0.1
0.07% of the incoming total nitrogen load) in this study seem to be the same range as those reported in the literature. In a lab-scale partial nitrification system, 5.4% of converted nitrogen was emitted as N2O at a DO level of 1.0 mgO2 L1 (Zheng et al., 1994). In a lab-scale nitrifying airlift reactor operated at DO concentration below 0.032 mg-O2 L1, 5.5% of the consumed ammonium was emitted as N2O (Sliekers et al., 2005). The emissions from lab-scale anammox

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Fig. 3 e Average nitrogen mass balance and N2O emission from the partial nitrification and anammox reactor during the measurement period (only the data after 33 days were used for the calculation). Numbers are average nitrogen loads (in mgN dL1) of influent and effluent of the PN and anammox reactor. N2O concentrations were measured in the liquid and off-gas. The off-gas stream of anammox reactor was created by the gas production. Percentages are relative to the nitrogen load of the partial nitrification reactor.

enrichment reactors were 0.03e0.06% (Strous et al., 1998), <0.1% (van de Graaf et al., 1997; Wyffels et al., 2004), and <0.01% (Kampschreur et al., 2008). Emission of N2O from a partial nitrification reactor treating the anaerobically treated concentrated black water at 25  C was 0.6e2.6% (average 1.9%) of the total nitrogen load (de Graaff et al., 2010). Furthermore, emission of N2O from a full-scale two-reactor partial nitrificationeanammox process treating reject water was determined to be 2.3% of the total nitrogen load (1.7% in the partial nitrification process and 0.6% in the anammox process) (Kampschreur et al., 2008). Emission of N2O from a full-scale single-stage partial nitrificationeanammox reactor treating wastewater from a potato processing factory and reject water of a municipal sludge dewatering plant was 1.2% of the total nitrogen load to the reactor (Kampschreur et al., 2009b). However, emission of N2O from a lab-scale single reactor partial nitrificationeanammox system on artificial wastewater was less than 0.1% of the nitrogen load (Sliekers et al., 2002). Moreover, Ahn et al., (2010) have surveyed several fullscale conventional nitrogen removal processes and found that 0.01e3.3% of influent total nitrogen was converted to N2O. Thus, the N2O emission level observed in this study is similar to other reported cases. In the PN reactor, since both ammonium and nitrite were in excess of oxygen (i.e., oxygen-limited condition), it is suspected that ammonium-oxidizing bacteria (AOB) outcompeted nitrite-oxidizing bacteria (NOB) (Okabe et al., 1996; Kindaichi et al., 2006) and produced N2O during denitrification of nitrite with ammonium as electron donor (Colliver and Stephenson, 2000). Oxygen is the most influential

factor affecting the production of N2O; a decrease in oxygen can result in activation of nitrite reductase and a severalfold increase in N2O production (Colliver and Stephenson, 2000).

3.3. NHþ 4 and N2O concentration profiles in anammox granules Even though N2O emission from the anammox rector was low, the steady-state concentration profiles of N2O and NHþ 4 in the anammox granules were determined with microelectrodes to study the mechanism of N2O emission. The concentration profiles of N2O and NHþ 4 were measured at least three times, and the average profiles are presented in Fig. 4A. The spatial distributions of net volumetric NHþ 4 consumption and N2O production rates were calculated based on the NHþ 4 and N2O concentration profiles (Fig. 4B and C). The Oxygen concentration was under the detection limit (ca. 0.3 mM) throughout the granules at all points. The concentration profiles indicated that NHþ 4 consumption by anammox reaction was restricted to the upper 1200 mm of the anammox granule with a peak around the upper 300 mm (Fig. 4B), while N2O production was found within a depth of 600e1300 mm with a peak around 800 mm (Fig. 4C). These results indicate that the anammox activity and N2O production are spatially separated, which probably suggests that the ammonium oxidation by anammox bacteria could not be the major biological process that is responsible for N2O production. This result does not completely negate the contribution of anammox bacteria to N2O emission, and thus further study is essential.

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 4 6 1 e6 4 7 0

40

were also detected around the anammox bacteria, and its relative abundance increased to 15e30% of the total bacteria hybridized with EUB mixed probe (EUB338, EUB338II and EUB338III) with the granule depth (Fig. 5E). The concentration profile of N2O in the anammox granules has neither been determined with microelectrode nor related to microbial community structure so far. Since active N2O production was detected in the deeper part of granule where aerobic AOB was not detected, the contribution of AOB (i.e., nitrifier denitrification) to the N2O production was negligible. Furthermore, there are no indications of N2O production by anammox bacteria so far, it seems that anammox bacteria are not responsible for N2O production. Taken together, the bacteria present in the depth of granules are most likely putative heterotrophic denitrifying bacteria and responsible for N2O emission in anammox granules.

30

3.4.

20

In order to examine the effect of pH on N2O emission in the anammox granules, the concentration profiles of N2O in the anammox granules were determined with microelectrodes at pH 7.0 and 8.0 (Fig. 6). The N2O production was higher at pH 7.0 than at pH 8.0. Batch culture experiments at different pH also reveal that the N2O production at pH 6.5 was about 11 times higher than pH 8.0 (Fig. 7). This pH dependent N2O production is probably attributed to inhibition of N2O reductase at low pH (Knowles, 1982). Actually, N2O reductase of heterotrophic denitrifiers is strongly inhibited by free nitrous acid (HNO2). A HNO2 concentration of 0.004 mg HNO2-N L1 completely inhibited N2O reduction during denitrification (Zhou et al., 2008). The medium used for microelectrode measurement contained 4.5 mM (63 mg-N L1) of NO 2 , corresponding to about 0.015 mg HNO2-N L1 at pH 7 and 0.0013 mg HNO2-N L1 at pH 8, respectively. Thus, it could be expected complete inhibition of N2O reductase at pH 7 or 6.5. Similar result has been reported by Hanaki et al. (1990), where N2O emission during heterotrophic denitrification increased when the pH decreased 8.5e6.5. Thoern and Soerensson (1996) have also reported that significant N2O formation was observed only at pH 6.8 in a denitrification basin. If N2O were produced via nitrifier denitrification, N2O emission could increase when pH increases (Kampschreur et al., 2009a,b). The pH dependent N2O emission in this study supports our conclusion that heterotrophic denitrification is most likely the main biological mechanism of N2O emission in anammox granules. In order to identify the source of N2O production in the anammox granule, we further determined N2O emission from anammox granules cultured in the medium supplemented   þ  with; (1) NHþ 4 and NO2 , (2) NO2 only, and (3) NH4 , NO2 , and penicillin G (Fig. 8). In the anoxic batch culture supplemented  with NHþ 4 and NO2 , AOB, anammox bacteria, and heterotrophic denitrifiers are supposed to be active. In the anoxic batch cultures supplemented with NO 2 , only heterotrophic denitrifiers are supposed to be active. Furthermore, in the anoxic  batch cultures supplemented with NHþ 4 , NO2 , and penicillin G, only anammox bacteria are supposed to be active because penicillin G inhibits most of the heterotrophic denitrifiers and AOB (van de Graaf et al., 1996; Gu¨ven et al., 2005). N2O production in the batch culture supplemented with NO 2 only

100

5000

80

4000 NH4+

3000

60

N2O

40

2000

20

1000

N2O concentration (µM)

NH4 + concentration (µM)

A

biofilm 0

0

Biofilm depth (µm)

B NH4+ consumption rate (µmol cm-3 h-1)

50

10 0 -1000

N2O production rate (µmol cm-3 h-1)

C

6467

-500

0 500 1000 Biofilm depth (µm)

1500

4.0

3.0

2.0

1.0

0.0 -1000

-500

0 500 1000 Biofilm depth (µm)

1500

Fig. 4 e (A) The steady-state concentration profiles of NHD 4 and N2O in the anammox granule. The surface of the granule is at a depth of 0 mm. The points are measured means ± standard deviations (n [ 3), and the solid lines are the best fits from the model to calculate the volumetric consumption rate of NHD 4 and production rate of N2O. Spatial distributions of the estimated volumetric consumption rate of NHD 4 (B) and production rate of N2O (C).

The microbial community structure in the anammox granule was analyzed by FISH (Fig. 5A and B). The FISH images of cross-section of the granules showed that the anammox bacteria hybridized with the probe Amx820 were present throughout the granules (Fig. 5C). The relative abundance of the anammox bacteria was more than 90% of the total bacteria hybridized with EUB mixed probe (EUB338, EUB338II and EUB338III). Aerobic AOB hybridized with the probe Nse1472 were detected only in the granule surface and around the clusters of anammox bacteria (Fig. 5D). The bacteria that were not hybridized with the probes Amx820, Nso190 and Nsv443

Effect of pH on N2O emission in anammox granules

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w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 4 6 1 e6 4 7 0

Fig. 5 e (A) An enlarged photo of granular-sludge anammox reactor, (B) A anammox granule with a diameter of approximately 3 mm, (C) Confocal laser-scanning microscope image of thin cross-section of the anammox granule showing in situ spatial organization of anammox bacteria (yellow) and coexisting other bacteria (green) after fluorescence in situ hybridization with FITC-labeled EUB338mix probe and TRITC-labeled probe Amx820, (D), Coexistence of FITC-labeled Nse1472 probe-hybridized Nitrosomonas eutropha/europaea-like AOB (green) and TRITC-labeled Amx820 probe-hybridized anammox bacteria (red) in the surface of granule, and (E) Coexistence of FITC-labeled EUB338mix probe hybridized other bacteria (green) and TRITC-labeled Amx820 probe-hybridized anammox bacteria (yellow) in the inner part of granule. All bars on the images indicate 20 mm.

250 N2 O concentration (µM)

pH 7 200

pH 8

150 100

autotrophic reactor, leading to N2O emission due to incomplete denitrification. Another possibility is endogenous denitrification (i.e., utilization of internal storage compound such as poly-b-hydroxybutyrate (PHB)) (Kampschreur et al., 2009a). However, the carbon source for heterotrophic denitrification is presently unknown. The result of this study is not consistent with the literature showing that denitrification by AOB was considered the most probable cause of N2O production (0.6% of the nitrogen load) in a full-scale anammox reactor treating sludge reject water (Kampschreur et al., 2008). Thus,

(g-N2O emitted / g-N-consumed)

(263
39.2 ppm, n ¼ 3) was higher than that in the culture  supplemented with NHþ 4 and NO2 (148
10.0 ppm, n ¼ 3) (Fig. 8). No detectable N2O production was observed in the  culture supplemented with NHþ 4 , NO2 , and penicillin G. Based on the results obtained in this study, it is conceivable that heterotrophic denitrification could be a main process of N2O emission in the anammox granule. In this case, N2O is produced as an intermediate of incomplete heterotrophic denitrification due to low COD/N ratio. The putative heterotrophic denitrifying bacteria could use organic matter liberated by anaerobic degradation of biomass inside granules. However, biodegradable organic matter is limited in such

0.012

* 0.01 0.008 0.006 0.004 0.002 0

50 biofilm 0 Biofilm depth (µm)

Fig. 6 e The concentration profiles of N2O at pH [ 7.0 and 8.0 in the anammox granule. The surface of the granule is at a depth of 0 mm. The points are measured means ± standard deviations (n [ 3).

*

NC

pH6.5

pH7

pH8

Fig. 7 e The pH effect on N2O production in anammox granules cultured at pH of 6.5, 7.0 and 8.0, respectively. The N2O emission was expressed as g-N2O emitted per g-N consumed. The error bars indicate standard deviations of triplicate measurements (n [ 3). NC: negative control (the autoclaved biomass was incubated in the same medium at pH 8). P values were determined using the Student’s t-test. *, P < 0.05. N.D, not detected.

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 4 6 1 e6 4 7 0

N2 O concentration (ppm)

350

Core Research of Evolutional Science & Technology (CREST) for “Innovative Technology and System for Sustainable Water Use” from Japan Science and Technology Agency (JST).

*

300 250

200

Appendix. Supplementary material

150

100

Supplementary data related to this article can be found online at doi:10.1016/j.watres.2011.09.040.

50 0

ND NH4+ + NO 2 -

NO2-

NH4+ + NO 2+penicillin G

Fig. 8 e The production of N2O in the anammox granules cultured in the medium supplemented with; (1) NHD 4 and L D L NOL 2 , (2) NO2 only, and (3) NH4 , NO2 , and penicillin G. The error bars indicate standard deviations of triplicate measurements (n [ 3). P values were determined using the Student’s t-test. *, P < 0.05. N.D, not detected.

further research using a dual-isotope (18Oe15N) labeling technique (Wrage et al., 2005) is required to distinguish between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification in anammox granules.

4.

6469

Conclusions

A lab-scale two-reactor partial nitrificationeanammox process was developed to investigate N2O emission from both processes.  The average emission of N2O from the lab-scale partial nitrification and anammox process was 4.0
1.5% (9.6
3.2% of the removed nitrogen) and 0.1
0.07% (0.14
0.09% of the removed nitrogen) of the incoming nitrogen load, respectively.  The total amount of N2O emission from the PN reactor was correlated to nitrite (NO 2 ) concentration in the PN effluent rather than DO concentration.  The active N2O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N2O production zone.  Based on all experimental results (including microelectrode, FISH, and batch experiments with an inhibitor) obtained, the N2O emission in the anammox reactor (i.e., granules) is most likely originated from heterotrophic denitrification.

Acknowledgments This research was financially supported by Grant-in-Aid for the “Development of High-efficiency Biological Wastewater Treatment Technology Using Artificially Designed Microbial Communities” Project from the New Energy and Industrial Technology Development Organization (NEDO), Japan and by

references

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