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Presence and activity of anammox and denitrification process in low ammonium-fed bioreactors


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Bioresource Technology xxx (2006) xxx–xxx

Presence and activity of anammox and denitri?cation process in low ammonium-fed bioreactors
Bipin K. Pathak
a

a,*

, Futaba Kazama a, Yuko Saiki b, Tatsuo Sumino

c

Department of Ecosocial System Engineering, Kazama Laboratory, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi, 400-8511, Japan b Fundamental Research Laboratory, Asahi Breweries Ltd., Moriya-Shi, Ibaraki 302-0106, Japan c Matsudo Research Laboratory, Hitachi Plant Engineering and Construction Co. Ltd., Kamihongo 537, Matusdo, Chiba 271-0064, Japan Received 8 May 2006; received in revised form 29 August 2006; accepted 30 August 2006

Abstract A combination of anammox and denitri?cation process was studied for 300 days in low ammonium-fed bioreactors under the support of organic carbon. Nutrient pro?les, 15N-labelling techniques and qualitative ?uorescence in situ hybridization (FISH) probes were used to con?rm the nitrogen removal pathways and intercompetition among di?erent bacteria populations. About 80% of nitrogen removal was achieved throughout the study period. The results con?rmed that anammox bacteria were absent in the bioreactor inoculated with anaerobic granules only but they were present and active in the central anoxic parts of biopellets in the bioreactor inoculated with mixed microbial consortium from activated sludge and anaerobic granules. It also showed that the anammox bacteria were successfully enriched in the low ammonium-fed bioreactors. Results of this study clearly demonstrated that anammox and denitri?cation processes could coexist in same environment and anammox bacteria were less competitive than denitrifying bacteria. ? 2006 Elsevier Ltd. All rights reserved.
Keywords: Anammox; Anaerobic granules; Denitri?cation; Immobilized microbial consortium

1. Introduction Recently, the hydrosphere has become the main sink for excess nitrogen as a result of human activity (Galloway et al., 2003). Nitrogen in its various forms can deplete dissolved oxygen (DO) levels in receiving waters, stimulate aquatic growth, exhibit toxicity towards aquatic life, present a public health hazard and a?ect the suitability of wastewater for reuse (Smith, 2003; Wolfe and Patz, 2002). Nitrogen removal is one of the costly elements in wastewater treatments and simultaneous bionitri?cation and biodenitri?cation (Hsieh et al., 2003; Khin and Annachhatre, 2004; Ruiz et al., 2006) have been commonly used as the main processes to remove nitrogen from water and wastewater. The addition of external carbon source to

*

Corresponding author. Fax: +81 55 220 8193. E-mail address: bipinpathak@yahoo.com (B.K. Pathak).

the anoxic zone of treatment plants facilitates denitri?cation process. For a long time, this combination was considered the only way to remove ammonium from wastewater. More recently, shortcut processes by replacing nitrate with nitrite (van Dongen et al., 2001), or application of the anaerobic oxidation of ammonia (anammox) process (Jetten et al., 2005) have been considered as novel nitrogen removal process. The anammox process has recently been used extensively in ammonium-rich wastewater (Guven et al., 2004; Jetten et al., 2005; Third et al., 2005) and has also investigated in natural water (Kuypers et al., 2003). However, application of the anammox process in low ammonium nitrogen has been still limited. In the anammox process, ammonium is oxidized anaerobically to N2 by autotrophic bacteria using nitrite as the electron acceptor. According to Van de Graaf et al. (1996), the anammox process produces nitrate which is about 10% of the in?uent ammonium. This means that the anammox process removes only 90% of the incoming nitrogen leaving behind

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10% in the e?uent. In order to meet stringent e?uent standards, an improved process has to be explored. Although the control over the physiological requirements is crucial because of the involvement of two di?erent groups of bacteria (autotrophic and heterotrophic), a combination of both anammox and denitri?cation would give the complete solution to nitrogen removal. This combined method o?ers signi?cant economical advantages such as reduction in energy and low carbon substrate. Therefore, the aim of this work was to examine the simultaneous presence and activity of anammox and denitri?cation processes in low ammonia-fed single bioreactors under the support of external carbon substrates. 2. Methods 2.1. Biopellets fabrication Biomass from the activated sludge of a domestic wastewater treatment plant and anaerobic granules from fullscale up?ow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Ibaraki, Japan were collected separately. The collected activated sludge was entrapped in polyethylene glycol (PEG) prepolymer gel as biopellets (Pathak et al., 2005). The composition of the immobilized material was 10% (w/v) PEG prepolymer and 2% (w/v) activated sludge. The ?nal size of the cubic biopellets was 3 mm. 2.2. Reactor setup and operation Two up?ow bioreactors, namely AG (anaerobic granules) and BAG (biopellets and anaerobic granules) were designed such that each had an e?ective volume of 100 mL. The AG bioreactor was inoculated with anaerobic granules to 50% of the total volume bioreactor and the BAG bioreactor was inoculated with a mixture of biopellets and anaerobic granules to 20% of the total volume of bioreactor. A 20 g aliquot of caprolactone [(C6H10O2)n] (solid biodegradable plastic), equivalent to 30% of the e?ective volume of the bioreactor was supplied to the BAG bioreactor as an additional carbon source. Water samples with total nitrogen content of 1.47 ± 1.00 mg/L (>95% in the form of nitrate nitrogen) were retrieved from Shiokawa Reservoir (Yamanashi, Japan). Ammonium nitrogen and nitrite nitrogen were added to understand the nitri?cation and denitri?cation processes more precisely under oxygen limiting conditions. The modi?ed water was supplied to the both bioreactors continuously at the rate of 200 mL/day using peristaltic pumps. All experiments were performed for 300 days at 20 °C in temperature-controlled bioreactors (Eyelatron FL1-301NH, Japan) under 12 h hydraulic retention time (HRT). Nutrient pro?les, 15N-labelling techniques and ?uorescently labelled rRNA probes were used to identify the nitrogen removal pathway. The details of these methods are described in Schmid et al. (2005).

2.3. Nutrient analysis Grab samples of in?uents and e?uents were collected twice a week from all bioreactors for chemical analyses. Samples were ?ltered using Whatman GF/F ?lters (0.45 lm) and ammonium nitrogen (NH? –N), nitrate 4 nitrogen (NO? –N) and nitrite nitrogen (NO? –N) concen3 2 trations were measured colorimetrically as described in APHA (1998). Nitri?cation performance was calculated based on the change in ammonium concentration in the in?uents and e?uents with respect to the initial NH? –N 4 concentration. Summation of the di?erences in NO? –N 3 and NO? –N in the in?uents and e?uents was used to 2 determine the extent of denitri?cation. The nitrogen removal e?ciency was calculated as (Nin ? Nout), where Nout and Nin are the nitrogen concentrations (mg/L) in the e?uent and in?uent, respectively. The nitrogen removal rate (g-N/m3 day) was calculated as daily nitrogen load removed [daily ?ow rate · (Nin ? Nout)] per unit volume of reactors. Dissolved organic carbon (DOC) concentration in the e?uent was measured using a total organic carbon analyser (Shimadzu – TOC-VCSH, Japan). Although DOC inside the reactors and in e?uent could be slightly di?erent, the expected concentration was presumed closely similar and comparable. 2.4.
15

N-labelling techniques

To identify the nitrogen removal pathway in the bioreactors, water samples retrieved from the reservoirs were modi?ed by adding equal concentrations of 99% 15 NO? –N (Cambridge Isotope Laboratories, USA) and 2 native NH? –N. The modi?ed water sample was applied 4 as in?uent for experimental time of 266–296 days. The gas produced in the bioreactor was carefully collected using a syringe and transferred to a bottle containing saturated NaCl. The bottle was carefully ?lled with saturated NaCl to prevent sample contamination by atmospheric gases and any possible bubble entrapment. The bottle was sealed with thick butyl rubber stoppers and kept in refrigerator in an inverted position until delivery to the laboratory at Shoko Co. Ltd. (Saitama, Japan) for isotope analysis. The concentration of 14N14N, 14N15N and 15N15N were determined using a Hitachi RM1-2 mass spectrometer using atmospheric air was as standard. The experiments were performed in triplicate. 2.5. Microbial analysis using ?orescence in situ hybridization (FISH) Granules samples from the AG bioreactor as well as biopellet and granule samples from the BAG bioreactor were collected on 300 days of experiment. The oligonucleotide probes for domain bacteria (EUB338/EUB338II/ EUB338III), archea (ARC915), anammox bacteria (AMX820) and planctomycete (PCL46) were used as described by Daims et al. (1999), Stahl and Amann

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(1991), Schmid et al. (2000) and Neef et al. (1998), respectively. The combination of all EUB338/EUB338II/ EUB338III probes was mixed in equal proportion with ARC915, AMX820, and PLA46 separately in order to compare the targeted bacteria with background bacteria. All of the probes were complementary to regions of the small subunit of 16s rRNA molecules and were conjugated with ?uorescent dyes, ?uorescein isothiocyanate (FITC) and Cy3. The synthesized and puri?ed probes were purchased as Cy3 and FITC-labelled derivatives from Takara Bio Inc. (Shiga, Japan). Granules and biopellet sample ?xation, slice preparation and hybridization were performed as reported by Saiki et al. (2002). Hybridized slices were viewed immediately under an Olympus FLUOVIEW FV300 laser microscope equipped with an Ar/HeNe laser unit and IX70 microscope (Olympus, Tokyo). 3. Results and discussion The measured in?uent concentration of NH? –N, 4 NO? –N and NO? –N in both bioreactors was 2.3 ± 0.38, 3 2 1.47 ± 1.00 and 2.1 ± 0.63 mg/L, respectively. Average pH in both bioreactors remained at 6.7 ± 0.8 but the DO in the in?uents varied from 0.5 to 4.0 mg/L. 3.1. Nitrogen removal activities in the AG bioreactor Nitrogen mass balance showed that the average nitri?cation and denitri?cation e?ciencies were 36% and 85%, respectively. The average nitrogen removal e?ciency was 60% with average nitrogen removal rate of 6.9 ± 1.6 g-N/ m3 day. However, the extent of removal was di?erent for di?erent N-forms. For example, the average NO? –N and 2 NO? –N removal was 93 ± 20% and 64 ± 25%, respec3 tively. But NH? –N removal was incomplete (36 ± 28%), 4 and was ?uctuating over the study period (Fig. 1). The NH? –N reduction was not proportional to the reduction 4 of NO? –N and somewhat lower than expected. The aver2 age ratio of ?NO? –N?=?NH? –N? conversion was 1.98 4 2 (Fig. 2), which was higher than stoichiometric ratio. The ?uctuation in nitrogen removal was probably due to microbial diversity and the physical properties of anaerobic
4 Concentration (mg/L) 3 2 1 0 0 50 100 150 200 Operation (days) 250 300

Ratio of NO 2 -N/NH4 -N removed

6 5 4 3 2 1 0 0 50 100 150 200 Operations (days) 250 300

Fig. 2. Ratio of NO? –N and NH? –N converted in AG bioreactor (black 2 4 square) and BAG bioreactor (white circle). The dotted line indicates the ? ? optimal (NO2 –N?=?NH4 –N) ratio for anammox process according to Van de Graaf et al. (1996).

granules such as multilayer structure with channel and void areas (Diaz et al., 2006). As investigated by Diaz et al. (2006) and Saiki et al. (2002) ?rmicutes were the predominant bacteria in the anaerobic granules which were dominant denitri?ers (Heylen et al., 2006). In the present work, these denitri?ers could have out-compete the nitri?ers because of presence of relatively high DOC concentrations. Measured e?uent DOC concentration varied from 2 to 5 mg/L and results showed that ammonium removal was unfavourable when e?uent DOC greater than 3 mg/L (Pathak et al., 2005). Moreover, DOC could have produced from anaerobic granules as investigated and reviewed by Diaz et al. (2006), Hulsho? Pol et al. (2004) and Waki et al. (2004). 3.2. Nitrogen removal activities in the BAG bioreactor Nitrogen mass balance showed that the average nitrogen removal e?ciency was 80% and removal rate was 9.3 ± 1.7 g-N/m3/day. Nitri?cation and denitri?cation e?ciencies were 75% and 92%, respectively. The e?uent C/N ratio varied from 0.6 to 4.0. Monitoring of NO? –N con2 centrations showed that the NO? –N production was insig2 ni?cant and denitri?cation was not inhibited by NO? –N. 2 The NO? –N was almost completely removed (>97 ± 6%) 2 and NH? –N and NO? –N removal e?ciencies were 4 3 75 ± 21% and 67 ± 25%, respectively. It clearly showed that NO? –N removal e?ciency was lower than NH? –N 4 3 removal e?ciency. Moreover, e?uent NO? –N was always 3 less than in?uent nitrogen (Fig. 3) and it neither accumulated nor entirely eliminated despite its low in?uent concentration (1.47 ± 1.00 mg/L). Probably there could be other unknown sink of NH? –N. 4 In order to explore the loss of NH? –N, evidence for the 4 anammox process in the bioreactor was tested from reaction stoichiometry. The measured ratio of NO? –N to 2 NH? –N conversion was 1.29, which was very close to the 4 stoichiometric relationship between nitrite and ammonium in the anammox process (Van de Graaf et al., 1996) (Fig. 3). The ratio also agreed well with values obtained in large scale wastewater treatment (Guven et al., 2004).

Fig. 1. Nitrogen concentration in the AG bioreactor, e?uent NH? –N 4 (white circle), e?uent NO? –N (black square), e?uent NO? –N (white 3 2 diamond), average in?uent NH? –N (solid line), and average in?uent 4 NO? –N (dotted line). 2

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3 Concentrations (mg/L)

B.K. Pathak et al. / Bioresource Technology xxx (2006) xxx–xxx

2

1

0

0

50

100

150 200 Operation (days)

250

300

Fig. 3. Nitrogen concentration in BAG bioreactor, e?uent NH? –N 4 (white circle), e?uent NO? –N (black square), e?uent NO? –N (white 3 2 diamond), average in?uent NH? –N (solid line), and average in?uent 4 NO? –N (dotted line). 2

than in atmospheric air in both reactors (Table 1). However, the variation of 14N15N was high in the AG bioreactor and low in the BAG bioreactor. On the other hand, production of 15N15N was the result of coupling of 15 15 NO? –N= NO? –N via denitri?cation. This showed that 2 2 the denitri?cation e?ciency was two times higher in the AG bioreactor than that in BAG bioreactor. Thus, isotope labelling technique clearly showed that both bioreactors were suitable for denitri?cation. But the possibility of anammox process to occur appeared only in the BAG bioreactor. Therefore, molecular analysis technique, such as FISH, was used to con?rm the existence of anammox bacteria. 3.4. Distribution of microorganisms in biopellets and granules The characterization of the microbial community in the bioreactors was examined using FISH technique. Hybridizing cells from the AG bioreactor with the FISH probes ARC915 and EUB338 together, produced a strong and weak signals (pictures not shown here), respectively. Methanogens were very active and colonies were abundant at or near granules surface. The domain bacterial colonies were scatted along the surface and central part of the granules (results not shown). Similar results of domain bacterial and methanogens population were obtained before (Saiki et al., 2002). In addition, cells hybridization with the FISH probes AMX820 for Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis; and PLA46 for planctomycetes were negative and no ?uorescent signal was detected. It was possible that abundant methanotrophs in the AG bioreactor could have oxidised methane and produced methanol (Waki et al., 2004), thereby inhibited the anammox process (Guven et al., 2005). No signal was detected when granule slices taken from the BAG bioreactor were hybridized with the ARC915 probe. It was probably due to low oxidation reduction potential (ORP) inside the BAG bioreactor, which inhibited the growth of methanogens. Cell hybridization with FISH probe PLA46 showed weak signal, indicating presence of lower planctomycetes populations. Furthermore, hybridizing cells of the biopellets from the BAG bioreactor with AMX820 and EUB338 produced a bright signal. Both anammox and domain bacteria were active in biopellets. These results suggested that anammox bacteria primarily grew at the central part of biopellets (200 lm from the surface) but at low population. Similarly, domain bacteria were distributed in outer layer of biopellets with abundant population. This result indicated that size distribution and layer thickness of biopellets a?ected the steady state oxygen concentration (Nielsen et al., 2005) and inhibited the growth of anammox bacteria on the surface (Third et al., 2005). This result was also consistent with Nielsen et al. (2005), where maximum anammox capacity was observed in the central anoxic part for aggregate size greater than 500 lm. The size of the biopellet e?ectively controlled the

Despite the stoichiometry ratio well agreed with anammox process, this ratio could be similar in normal denitri?cation (Dalsgaard et al., 2003), hence, required further veri?cations using conventional techniques. The total NH? –N 4 reduction was not proportional to the reduction of NO? –N and much more NO? –N than required for the oxi2 2 dization of ammonium was consumed. It meant that fractions of total NO? –N could be reduced through 2 denitri?cation using organic compounds as electron donors supported by the average C/N of 0.6 (Cervantes et al., 2001). On the other hand, the mixed bacterial consortium was capable for almost complete removal of 8–10 g-N/ m3 day at 12 h HRT. Consistent results were achieved in Foglar et al. (2005), where selected mixed microbial consortium originating from an industrial wastewater treatment plant was suggested more advantageous than pure culture for fast denitri?cation without nitrite accumulation. 3.3.
15

N-labelling techniques

In order to trace the nitrogen removal pathway, the bioreactors were fed with 15 NO? –N and native NH? –N. 2 4 Anammox produced 14N15N and 15N15N whereas denitri?cation produced 14N14N, 14N15N and 15N15N through random isotope pairing when unlabelled 14N and labelled 15N were applied (Dalsgaard et al., 2003). Because the anammox process combines 1 mole of (15N) nitrite and 1 mole of (14N) ammonium to form 1 mole of single-labelled dinitrogen gas (14N15N), the isotope fraction of 14N15N with reference to atmospheric air expresses the anammox potential activity. The 14N15N observed in this study was higher
Table 1 Isotope fractions of N2 (%) with mass 28 (14N14N), 29 (14N15N) and 30 (15N15N) measured in the AG and BAG bioreactors after the addition of 15 NO? –N compounds, compared with reference atmospheric N2 2 Bioreactors AG BAG Standard (air)
14

N14N (%)

14

N15N (%)

15

N15N (%)

96.96 ± 1.44 98.35 ± 0.25 99.16 ± 0.15

2.07 ± 0.83 1.25 ± 0.14 0.83 ± 0.10

0.98 ± 0.61 0.40 ± 0.12 0.01 ± 0.00

The results are mean values of three replicate (n = 3) experiments.

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population distribution such as larger size aggregates promoted the anammox activity whereas smaller size promoted the nitri?cation potential (Nielsen et al., 2005). However, the present study was unable to ?nd the exact thickness of biopellets where anammox activity was highest. These results suggested that the denitrifying bacteria could out-compete anammox bacteria, especially in an organic carbon rich environment. This was likely due to low activity and growth rate of anammox bacteria which populated together with predominantly by fast growing heterotrophic denitri?ers. Similar results of coexistence of anammox and denitri?cation process was found by Dong and Tollner (2003) in anaerobic digestion of poultry manure. These results collectively suggested that anammox was less competitive than denitri?cation. 4. Conclusions A mixture of an immobilized microbial consortium of activated sludge and anaerobic granules exhibited complete nitrogen removal through the combination of anammox and denitri?cation processes than single source of microbial consortium. Results demonstrated that both anammox and denitri?cation processes worked symbiotically in low ammonium concentrations under the support of external organic matters. These results showed the feasibility for the future implementations of anammox process water and wastewater consisting of low ammonium concentration in the presence of organic matters. Acknowledgements This work was supported by University of Yamanashi, 21st Centaury, Center of Excellence, Japan. We thank Yukiko Hiraga for technical help and Deb Jaisi for critical comments on the manuscript. References
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