Biochemical Engineering Journal 41 (2008) 59–66

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Biochemical Engineering Journal

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Nitrogen-removal bioreactor capable of simultaneous nitrification anddenitrification for application to industrial wastewater treatment

Masahiko Morita, Hiroaki Uemoto ∗, Atsushi Watanaber Indus

0 pacontaiosomolectror was, andal ratthanoainedessarthe sld beneou

Environmental Science Research Laboratory, Central Research Institute of Electric Powe

a r t i c l e i n f o

Article history:Received 30 August 2007Received in revised form 1 January 2008Accepted 17 March 2008

Keywords:Nitrogen removalWastewater treatmentBioreactorImmobilized bacteriaNitrificationDenitrification

a b s t r a c t

A bioreactor system with 3nitrogen from ammonia-cplate gels containing Nitrbetween for injecting an estep. When the wastewatetotal nitrogen in the inletmaximum nitrogen removefficiency of the injected ein the outflow was mainteffectively, it was not necsettling tank to segregatepacked gel envelopes wousystem capable of simulta

1. Introduction

Eutrophication, which is caused by an excessive nitrogen inflowfrom domestic and industrial effluents, causes severe adverseeffects on the environment of closed water system [1]. In Japan,algal blooms and red tides occur frequently and have caused dam-age to the water service and fishing industry in Tokyo Bay, Ise Bay,and the Seto Inland Sea. Therefore, strict regulations were appliedfor discharging nitrogen effluents into the industrial wastewater.

The ammonia removal process using microorganisms has nowbeen divided into two kinds of biological processes: one is thetraditional nitrification/denitrification method [2], and the otheris a newly developed anaerobic ammonium oxidation (anammox)method [3,4]. The anammox process combines ammonia and nitriteinto nitrogen gas anaerobically. It is an attractive method withgreat promise. However, the anammox process requires an addi-tional nitrification process for the partial conversion of ammoniato nitrite.

Presently, nitrogen (ammonia) removal is mostly carried outthrough two conversion steps, namely, aerobic nitrification andanaerobic denitrification. This method, however, has a disad-vantage. When treating wastewater without organic matters, an

∗ Corresponding author. Tel.: +81 4 7182 1181; fax: +81 4 7183 3347.E-mail address: uemoto@criepi.denken.or.jp (H. Uemoto).

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bej.2008.03.008

try (CRIEPI), 1646 Abiko, Abiko-shi, Chiba 270-1194, Japan

ked gel envelopes was installed in a thermal power plant for the removal ofning desulfurization wastewater. Each envelope consisted of double-sidednas europaea and Paracoccus denitrificans cells with an internal space inn donor. The envelope can remove ammonia from wastewater in a singlecontinuously treated with the bioreactor system, it removed 95.0% of the

the total nitrogen concentration in the outlet was below 9.0 mg L−1. Thee was 6.0 g day−1 per square meter of the gel area. The maximum utilizationl for denitrification was 98.4%, and the total organic carbon concentrationat a low level. Since the bioreactor system could use the electron donor

y to use an additional aerobic tank to remove the electron donor and aurplus sludge containing bacteria from wastewater. Our concept of usinghighly effective for constructing a simple and efficient nitrogen removal

s nitrification and denitrification.© 2008 Elsevier B.V. All rights reserved.

electron donor is required for denitrification. In this case, it requiresan additional aerobic step to remove surplus electron donors anda settling step to segregate the activated sludge containing bacte-ria from the wastewater. Since there are many steps in the presentnitrogen removal systems, a complicated sequence of operations is

necessary and a large installation area is required.

Several researches have been conducted in an attempt to com-bine the two conversion steps (nitrification and denitrification) intoa single bioreactor [5–7]. Polymeric beads, in which a nitrifier anda denitrifier were coimmobilized, were used to remove nitrogen ina single step. Nitrification occurred on the outer layer of beads, anddenitrification, in the core of beads. However, the electron donorswere added directly to the wastewater in these systems. Therefore,an additional aerobic step was required for the removal of surpluselectron donors, and the efficiency of utilizing electron donors wasrelatively low.

To simplify the present systems used for nitrogen removal, wehave proposed and investigated a novel immobilized-cell biore-actor containing packed gel envelopes capable of simultaneousnitrification and denitrification [8]. The packed gel envelopes con-sist of two polymeric gel plates with an internal space betweenthem for injecting the electron donor for denitrification. An ammo-nia oxidizer, namely, Nitrosomonas europaea, and a denitrifier,namely, Paracoccus denitrificans, are coimmobilized in the plate gel.The immobilized N. europaea oxidizes ammonia to nitrite on theouter surface of the plate that is in aerobic contact with the wastew-

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Nomenclature

A gel area of each envelope (m2)a absolute ethanol volume injected into each packed

gel envelope per day (mL day−1)b purity of used ethanolc nitrogen mass capable of denitrifying per ethanol

mass (g g−1)Rmax theoretical maximum nitrogen removal rate per

square meter of the gel area (g m−2 day−1)� relative density of ethanol at 20 ◦C (g mL−1)

ater containing ammonia; the immobilized P. denitrificans reducesnitrite to nitrogen gas in the inside of plate that is in anaerobiccontact with the electron donor. This system does not require anadditional aerobic step because the electron donors are not sup-plied to the wastewater directly but to the internal space of the gelplate. This results in an increase in the utilization efficiency of theelectron donor for the denitrification process and a decrease in thequantity of surplus sludge.

In this study, we investigated a large-scale bioreactor using thin-ner packed gel envelopes in which N. europaea and P. denitrificanscells are coimmobilized, treated the ammonia-containing wastew-ater from a coal-fired thermal power plant, and studied the nitrogenremoval capacity of the system.

2. Materials and methods

2.1. Bacterial strains and culture mediums

The nitrifier N. europaea NBRC 14298 and the denitrifier P.denitrificans JCM 6892 were used in this study. N. europaea wasaerobically cultured at 30 ◦C in a medium containing the following(g L−1): (NH4)2SO4, 0.5; NaCl, 0.3; K2HPO4, 1.0; MgSO4·7H2O, 0.3;FeSO4·7H2O, 0.03; phenol red, 0.002 (pH 8.0). P. denitrificans wasaerobically cultured at 30 ◦C in a medium containing the following(g L−1): peptone (Difco), 10.0; meat extract (Difco), 10.0; NaCl, 5.0(pH 7.2). All nutrients were dissolved in distilled water.

2.2. Characteristics of the actual wastewater

Desulfurization wastewater from the Takehara thermal power

plant (Takehara City, Hiroshima Prefecture, Japan) was used inthis study. The maximum output of this coal-fired thermal powerplant was 1300 MW. The heavy metals contained in this desul-furization wastewater were removed in a previous process. Theorganic carbon content of the wastewater was extremely low.The total organic carbon (TOC) concentration in the wastewa-ter ranged between 0.0 and 5.4 mg L−1. The form of nitrogen inthe wastewater was mostly ammonia (17.6–108.4 mg L−1). More-over, nitrite (3.8–37.3 mg L−1) and nitrate (0.0 to 26.6 mg L−1) wereoften detected during the operations. Phosphates were not detected(<0.1 mg L−1) in the wastewater although large amounts of sulfatewere observed (2412–13,176 mg L−1).

2.3. Packed gel envelope

N. europaea and P. denitrificans cells were harvested by cen-trifugation (20,000 g, 10 min, 4 ◦C) and washed 3 times withphosphate buffer containing 9.0 g L−1 of Na2HPO4·12H2O and1.5 g L−1 KH2PO4 (pH 7.5). N. europaea (dry weight, 4 mg mL−1) andP. denitrificans (dry weight, 3.4 mg mL−1) cells were suspended inphosphate buffer. The suspension was then mixed with the photo-

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crosslinkable polymer PVA-SbQ (SPP-H-13; Toyo Gosei Kogyo Co.)in the ratio of 1:3.

Each packed envelope (envelope I) (1100 mm × 1100 mm ×12 mm) consisted of a frame made of vinyl chloride, and nonwo-ven nets made of polyethylene terephthalate (G2260-1S; TorayCo.) were attached to both the aspects of the frame (Fig. 1a andb). The above mentioned bacteria–polymer mixture was spreadon the nonwoven net of the envelope and was gelled by irradi-ating with metal halide lamps for 20 min (1000 �mol m−2 s−1). Asthe bacteria–polymer mixture penetrated into the nonwoven netslightly, the gel was solidified on the nonwoven net. The heightand width of gel sheet were both 1000 mm. The thickness ofthe gel sheet on the net was 0.5 mm. Next, the same procedurewas followed for the opposite side of the envelope. As the enve-lope had two gel sheets and one-sided surface of each gel sheetcould be contacted with wastewater, the area of active regionwas 2.00 m2 in the envelope I. This value was used as unit gelarea for each envelope I. In addition, a thinner packed envelope(envelope II) (1250 mm × 1100 mm × 3.5 mm) was developed usingthe same nonwoven net without a frame (Fig. 1c and d). Thebacteria–polymer mixture was spread on the nonwoven net of openenvelope II, and was gelled by same way. The height and width of gelsheet were 1200 and 1050 mm, respectively. The thickness of thegel sheet was also 0.5 mm. After gelation, the nonwoven net immo-bilizing bacteria was folded as the gel sheet outside and made toan envelope. The area of active region was 2.52 m2 in the envelopeII. This value was used as unit gel area for each envelope II. Thesepacked gel envelopes contained internal spaces for injecting theelectron donor. Three holes were made at the top of each envelope,and polyurethane tubes (outer diameter, 8 mm; inner diameter,6 mm) were attached to the holes. Two holes were made as theoutlets for nitrogen gas after denitrification, and one central holewas made to inject the electron donor for denitrification.

2.4. Bioreactor system

A large-scale bioreactor system was installed in theTakehara thermal power plant. The system consisted ofa reactor tank (1.23 m × 1.13 m × 1.50 m); an overflow tank(0.29 m × 1.13 m × 1.50 m); a neutralization tank for pH adjust-ment; packed gel envelopes; pumps for wastewater circulation; ablower for aeration; a feeder for the electron donor; meters andrecorders for temperature, pH, and dissolved oxygen concentration

in the reactor tank; and a control panel. Fig. 2 shows the schematicdiagrams of the system during batch and continuous operations.

2.5. Batch operation

The batch operations were carried out overnight using 10 packedgel envelopes (envelope I) with frames containing N. europaea andP. denitrificans (Fig. 1a and b) under various ammonia concentra-tions by adding ammonium sulfate. Desulfurization wastewater(containing mostly ammonia, and some nitrite and nitrate) orig-inating from the thermal power plant was treated in the abovementioned bioreactor system (working volume, 2.3 m3). For 50 mLof 10% ethanol solution, phosphate (Na2HPO4·12H2O, 0.09 g L−1

and KH2PO4, 0.015 g L−1) and trace elements (MgSO4·7H2O,2 mg L−1; CaCl2·2H2O, 0.1 mg L−1; NaHCO3, 5 mg L−1; EDTA-Fe,0.05 mg L−1; ZnSO4·7H2O, 1 mg L−1; MnCl2·4H2O, 0.3 mg L−1;H3BO3, 3 mg L−1; CoCl2·6H2O, 2 mg L−1; CuCl2·2H2O, 0.1 mg L−1;NiCl2 6H2O, 0.2 mg L−1; and Na2MoO4·2H2O, 0.3 mg L−1) wereadded; this solution was injected into the internal space of eachenvelope every 6 h. The wastewater was sampled for analysis dur-ing the batch operation.

M. Morita et al. / Biochemical Engineering Journal 41 (2008) 59–66 61

Fig. 1. (a) Schematic diagram and (b) photograph of packed gel envelope (envelope I) with a frame made of vinyl chloride and nonwoven nets made of polyethyleneterephthalate. (c) Schematic diagram and (d) photograph of the thinner packed gel envelope (envelope II) using the same nonwoven nets without frame.

Fig. 2. Schematic flow diagrams of the system in (a) batch and (b) continuous operations.

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Fig. 3. Time-dependent changes in the concentrations of ammonia, nitrite, nitratetration: (a) 92.2 mg L−1; (b) 66.8 mg L−1.

2.6. Continuous operation

The wastewater was continuously treated using the developedbioreactor system (working volume, 1.8 m3) with 30 thinner packedenvelopes (envelope II) without frames (Fig. 1c and d) at the inflowrates of 0.075 (operation 1) or 0.45 (operation 2) m3 h−1 (aver-age residence time, 24 or 4 h) for nearly one month each. Every6 h, 40 mL of 10% ethanol solution was injected into each envelope(160 mL day−1 for each envelope). The wastewater was sampled foranalysis every 4 h from 9:00 a.m. to 9:00 p.m. A heater was used

for warming the wastewater during winter.

2.7. Analytical methods

The nitrogen concentrations were measured as NH3–N, NO2–N,and NO3–N. The ammonia concentration in the wastewater was col-orimetrically measured according to a previously described method[9]. The nitrite and nitrate concentrations were determined usingan ion-chromato analyzer (DX-AQ; Dionex Co.) with an IonPacAS12A column. The TOC concentration was measured using a TOCanalyzer (TOC-650; Toray Engineering Co.).

3. Results

3.1. Batch operation using ammonia-containing wastewater

When the wastewater containing various concentrations ofammonia was treated with 10 packed gel envelopes (envelope I)with frames containing N. europaea and P. denitrificans, the ammo-nia concentration in the wastewater decreased gradually (Fig. 3).A small amount of nitrate was detected (maximum, 2.6 mg L−1)

Fig. 4. (a) Relationship between initial ammonia concentration and ammonia oxidationnitrogen concentration and nitrogen removal rate (NH3 → N2) during batch operations.

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otal nitrogen in the wastewater during batch operations. Initial ammonia concen-

during all batch operations. As a result, the total nitrogen concen-tration (ammonia, nitrite, and nitrate) in the wastewater decreasedgradually. The TOC concentration in the wastewater was unchanged(average, 2.1 mg L−1). The pH in the wastewater ranged between 7.9and 8.4. The temperature in the wastewater ranged between 22.0and 29.0 ◦C.

The ammonia oxidation rate (the transformation of ammoniato nitrite) by 10 envelopes (envelope I) was calculated based onthe change in ammonia concentration every 2 h during batch oper-ations. The system yielded ammonia oxidation rates in the range

of 1.0–9.6 g day−1 per square meter of the gel area at various ini-tial concentrations of ammonia. Additionally, the nitrogen removalrate (the transformation of ammonia, nitrite, and nitrate to nitrogengas) was calculated based on the change in the total nitrogen con-centration every 2 h during batch operations. The system yieldednitrogen removal rates in the range of 1.2–19.5 g day−1 per squaremeter of the gel area. The relationship between the nitrogen con-centration and nitrogen removal rate during the batch operation isshown in Fig. 4. The rate of ammonia oxidation was proportional tothe ammonia concentration. Moreover, the rate of nitrogen removalwas proportional to the total nitrogen concentration. Hence, thehigher the total nitrogen concentration in the wastewater, thehigher was the nitrogen removal rate.

3.2. Nitrogen removal in continuous operation usingammonia-containing wastewater

The gel area of all envelopes per working volume has an effect onthe volumetric nitrogen removal performance. New thinner packedgel envelopes (envelope II) were developed using nonwoven netsmade of polyethylene terephthalate so as to increase the total num-

rate (NH3 → NO2−) during batch operations. (b) Relationship between initial total

M. Morita et al. / Biochemical Engineering Journal 41 (2008) 59–66 63

Fig. 5. Time-dependent changes in (a) nitrogen concentrations in the inflow andoutflow, (b) nitrogen removal rates and (c) nitrogen removal efficiencies during con-tinuous operation using 30 thinner envelopes (envelope II) at a wastewater flow rateof 0.075 m3 h−1 (operation 1). Symbols: ammonia concentration in the inflow (�)and outflow (�), nitrite concentration in the inflow (♦) and outflow (�), nitrate con-centration in the inflow (©) and outflow (�), and total nitrogen concentration in theinflow (�) and outflow (�) in (a); ammonia oxidation rate (�) and nitrogen removalrate (�) in (b); ammonia removal efficiency (�) and nitrogen removal efficiency (�)in (c).

ber of the packed gel envelopes capable of introducing into thereactor tank, namely, the gel area of all envelopes per workingvolume. Assuming that the nitrogen removal performance per gelarea of each envelope in the modified envelopes (envelope II) wasequivalent to that obtained in the original envelopes (envelope I),the volumetric nitrogen removal performance using the modified

envelopes was estimated to be twice as high as that using the orig-inal envelopes. Since the modified envelopes appeared promisingfor treating large volumes of wastewater, 30 modified envelopeswere used during the continuous operation.

The wastewater was continuously treated with 30 modifiedpacked gel envelopes (envelope II) for nearly one month each.The time-dependent changes in the nitrogen concentrations inthe inflow and outflow at the inflow rate of 0.075 m3 h−1 (oper-ation 1) are shown in Fig. 5a. The total nitrogen concentrationin the inflow (37.6–127.1 mg L−1) fluctuated abruptly as a resultof the sudden variation in the ammonia concentration in theinflow (17.6–108.4 mg L−1). Compared to the ammonia concentra-tion, the concentrations of nitrite (11.9–26.0 mg L−1) and nitrate(1.6–7.7 mg L−1) in the inflow did not fluctuate. The ammonia con-centration in the outflow was maintained at a low level (average,1.3 mg L−1) during the continuous operation. The concentrationsof nitrite (below 4.9 mg L−1) and nitrate (below 1.9 mg L−1) in theoutflow were also low. As a result, the total nitrogen concentra-tion in the outflow ranged between 0.4 and 9.0 mg L−1 (average,3.7 mg L−1). The time-dependent changes in the nitrogen removalrates and efficiencies at an inflow rate of 0.075 m3 h−1 (operation

Fig. 6. Time-dependent changes in (a) nitrogen concentrations in the inflow andoutflow, (b) nitrogen removal rates and (c) nitrogen removal efficiencies during con-tinuous operation using 30 thinner envelopes (envelope II) at a wastewater flow rateof 0.45 m3 h−1 (operation 2) Symbols: ammonia concentration in the inflow (�) andoutflow (�), nitrite concentration in the inflow (♦) and outflow (�), nitrate concen-tration in the inflow (©) and outflow (�), and total nitrogen concentration in theinflow (�) and outflow (�) in (a); ammonia oxidation rate (�) and nitrogen removalrate (�) in (b); ammonia removal efficiency (�) and nitrogen removal efficiency (�)in (c).

1) are shown in Fig. 5b and c. The rates of ammonia oxidation andnitrogen removal were calculated based on the differences betweenthe concentrations of ammonia and total nitrogen both in the inflowand in the outflow. The ammonia oxidation rate ranged between 0.4and 2.6 g day−1 per square meter of the gel area, and the nitrogenremoval rate was in the range 0.9–2.9 g day−1 per square meter ofthe gel area (Fig. 5b). The ammonia and nitrogen removal efficien-

cies, which were defined as the ratios of the differences betweenthe concentrations of ammonia and total nitrogen in the inflowand outflow to the concentrations of ammonia and total nitrogenin the inflow, were calculated from the concentrations of ammoniaand total nitrogen in the inflow and outflow. The bioreactor with30 modified envelopes (envelope II) removed most of the ammo-nia (average removal efficiency, 97.1%) and total nitrogen (averageremoval efficiency, 95.0%) in the wastewater (Fig. 5c). The aver-age TOC concentrations in the inflow and outflow were 0.4 and4.8 mg L−1, respectively. The pH in the wastewater ranged between7.2 and 8.4. The temperature in the wastewater was maintainedbetween 25.0 and 32.0 ◦C by using a heater. The results revealed thatthe modified envelopes could achieve high nitrogen removal effi-ciency. However, the maximum nitrogen removal rate at an inflowrate of 0.075 m3 h−1 was not very high, i.e., 2.9 g day−1 per squaremeter of the gel area; this was probably due to the low nitrogenload in the reactor tank.

The time-dependent changes in the nitrogen concentrations inthe inflow and outflow at an inflow rate of 0.45 m3 h−1 (operation 2)are shown in Fig. 6a. The total nitrogen concentration in the inflow(76.2–130.1 mg L−1) fluctuated abruptly due to the sudden variation

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in the ammonia concentration in the inflow, while the concen-trations of nitrite and nitrate in the inflow were unchanged. Theammonia, nitrite, and total nitrogen concentrations in the outflowvaried due to the abrupt fluctuation in the ammonia concentra-tion in the inflow (43.9–104.1 mg L−1). The nitrate concentrationin the outflow was unchanged. The time-dependent changes inthe nitrogen removal rates and efficiencies at the inflow rate of0.45 m3 h−1 (operation 2) are shown in Fig. 6b and c. The maxi-mum ammonia oxidation rate achieved was 9.5 g day−1 per squaremeter of the gel area. The bioreactor capable of simultaneous nitri-fication and denitrification achieved a maximum nitrogen removalrate of 6.0 g day−1 per square meter of the gel area. This maxi-mum rate obtained using the thinner envelopes was equivalentto that obtained with the original envelopes. During the first 5days, the average rates of ammonia oxidation and nitrogen removalwere 3.7 and 3.2 g day−1 per square meter of gel area, respectively.These rates increased to 4.9 and 3.5 g day−1 per square meter ofthe gel area in the next 5–10 days, and further increased to 7.1and 4.7 g day−1 per square meter of the gel area after 10 days. Theammonia and total nitrogen removal efficiencies ranged from 24.1%to 95.8% and from 13.5% to 45.0%, respectively. The average TOCconcentrations in the inflow and outflow were 1.6 and 0.9 mg L−1,respectively. The pH in the wastewater ranged between 7.3 and 8.5.The temperature in the wastewater was maintained between 25.0and 32.0 ◦C by using a heater.

4. Discussion

4.1. Nitrogen removal performance under batch operation

The bioreactor (working volume, 0.25 L) containing eight packedgel envelopes (height, 100 mm; length, 48 mm; thickness, 0.5 mm;gel area, 0.0096 m2) yielded a maximum ammonia oxidation rateand maximum nitrogen removal rate of 5.6 and 5.0 g day−1 persquare meter of the gel area, respectively, in the laboratory exper-iment [8]. The packed gel envelopes capable of simultaneousnitrification and denitrification should be scaled up in order to treatlarge amount of actual wastewater. Thus, the packed gel envelopescontaining N. europaea and P. denitrificans (height, 500 mm; length,1000 mm; thickness, 16 mm; gel area, 0.72 m2) were enlargedand the nitrogen removal performance was examined in the pre-liminary experiments. When the artificial ammonia-containingwastewater was treated using a laboratory batch system (work-

ing volume, 0.35 m3) with two packed gel envelopes, it exhibiteda maximum ammonia oxidation rate and maximum nitrogenremoval rate of 6.9 and 4.6 g day−1 per square meter of the gelarea, respectively (data not shown). Since the enlarged packed gelenvelopes achieved similar performance, packed gel envelopes offunctional size (envelope I) (height, 1100 mm; length, 1100 mm;thickness, 12 mm; gel area, 2.00 m2) were developed for thescaling-up of the system in order to treat large volume of theactual industrial wastewater. The maximum ammonia oxidationand nitrogen removal rates, which were calculated from the differ-ences in the ammonia and total nitrogen concentrations at the starttime and after 21.5 h, were 5.9 and 7.1 g day−1 per square meter ofthe gel area, respectively, during the batch operation. The large-scale system with the functional packed gel envelopes exhibitedhigh performance. Particularly, the nitrogen removal rate achievedin this research was higher than those obtained in other experi-ments, probably due to the presence not only ammonia but alsonitrite in the inlet wastewater.

The rates of ammonia oxidation and nitrogen removal wereproportional to the concentrations of ammonia and total nitro-gen, respectively. Because molecular oxygen was necessary for

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ammonia oxidation by N. europaea, nitrification was carried outon the outer surface of the gel [10]. In contrast, the denitrifica-tion by P. denitrificans was carried out in the inside of the gel sinceit required an anaerobic environment [10]. Thus, for nitrification,ammonia should be diffused near the outer surface of the packedgel envelopes. For denitrification, nitrite—which preexisted in thewastewater and was oxidized from ammonia by N. europaea on theouter surface of envelopes—and nitrate should be introduced intothe inner part of the packed gel envelope by diffusion. Because therate of diffusion of ammonia into the packed gel envelopes was pro-portional to the ammonia concentration, the ammonia oxidationrate was thought to be proportional to the ammonia concentra-tion. Further, the nitrogen removal performance appeared to beproportional to the total nitrogen concentration as the diffusion ofammonia, nitrite, and nitrate into the envelopes was proportionalto the total nitrogen concentration. This was caused by the diffusionof nitrogen into the envelopes. Thus, in this system, the diffusionof nitrogen into the packed gel envelope was one of the limitingfactors for nitrogen removal performance. The intensity of wastew-ater flow decreases the thickness of the interfacial film. This mayimprove the diffusion affecting the nitrogen removal performance.

4.2. Utilization efficiency of electron donor for denitrification

An electron donor (e.g., ethanol) is required for denitrification.The theoretical maximum rate of nitrogen removal per squaremeter of the gel area was calculated as follows:

Rmax = abc�

A(1)

where A is the gel area of each envelope (m2), a is the absoluteethanol volume injected into each envelope per day (mL day−1),b is the purity of ethanol used, c is the nitrogen mass capableof denitrifying per ethanol mass (g g−1), Rmax is the theoreticalmaximum nitrogen removal rate per square meter of the gel area(g m−2 day−1), and � is the relative density of ethanol at 20 ◦C(g mL−1). In this case, the values of A, b, and � were 2.52 m2, 0.995,and 0.789 g mL−1, respectively. In the continuous operation, since160 mL of a 10% ethanol solution was injected into each envelopeper day, the value of a was 16 mL day−1. The chemical reactionoccurring during the denitrification of nitrite by the use of ethanolis as follows:

4NO2− + C2H5OH → 2N2 + 2CO2 + H2O + 4OH− (2)

Thus, 1 mol of ethanol can denitrify 4.0 mol of nitrite, and thevalue of c was 1.22 g g−1. As a result, the theoretical maximum nitro-gen removal rate per square meter of the gel area was calculatedto be 6.1 g m−2 day−1. In this study, since the maximum nitrogenremoval rate achieved in the continuous condition was 6.0 g day−1

per square meter of the gel area, the maximum ethanol utilizationefficiency was 98.4%.

In the activated sludge system, the nitrite oxidized from ammo-nia by the ammonia-oxidizing bacteria was further oxidized tonitrate by the nitrite-oxidizing bacteria. The chemical reactionoccurring in the denitrification of nitrate by the use of ethanol isas follows:

12NO3− + 5C2H5OH → 6N2 + 10CO2 + 9H2O + 12OH− (3)

Thus, 1 mol of ethanol can denitrify only 2.4 mol of nitrate. Onthe other hand, in the present system, there was no nitrate accumu-lation in the wastewater since the denitrification of nitrite occurreddirectly. As a result, the ethanol requirement in the present systemwas theoretically 60% of that in the activated sludge system. In theactivated sludge system, the amount of electron donor empirically

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M. Morita et al. / Biochemical E

Table 1Volumetric nitrogen removal performances of the nitrogen removal systems using

Reactor system Polymeric gel fo

Water-jacketed glass column (Kokufuta et al. [7]) Calcium alginatpolyelectrolyte

Bubble column reactor (dos Santos et al. [5]) Calcium alginatReactor using immobilized pellet (Mori et al. [12]) Polyethylene glReactor using packed gel envelopes (Uemoto and Saiki [8]) Photo-crosslinkReactor using modified packed gel envelopes (this study) Photo-crosslink

injected for the denitrification step was twice that of the theoret-ical requirement. Thus, the present system can decrease ethanolrequirement to 30% as compared with the actual input.

In fact, the present system with the modified packed gelenvelopes (envelope II) could use the electron donor (e.g., ethanol)effectively in this study. The TOC concentration in the outflow wasmaintained at a low level (average concentrations were 4.8 and0.9 mg L−1 at hydraulic retention time of 24 and 4 h, respectively)through the continuous operation. On the other hand, in the anoxicdenitrification bioreactor containing packed gel envelopes in whichonly P. denitrificans cells were immobilized, a high level of TOC waspresent in the outflow when the total nitrogen concentration wasrelatively low [11]. This difference was caused by the installationof an aeration system in this pilot test, indicating that moderateaeration in the reactor tank is considerably effective in decreasingthe TOC concentration in the outflow. Thus, our bioreactor doesnot require an additional aerobic tank to remove surplus electrondonors released from the packed gel envelopes and a settling tankto segregate surplus sludge containing bacteria from the wastewa-ter. This concept of using the packed gel envelopes would be highlyeffective for constructing a simple and efficient nitrogen removalsystem capable of simultaneous nitrification and denitrification.

4.3. Volumetric nitrogen removal performance

The modified packed gel envelopes (envelope II) exhibited max-imum nitrogen removal rate of 6.0 g day−1 per square meter ofthe gel area. This value corresponded to the volumetric nitrogenremoval performance of 0.252 kg m−3 day−1. The volumetric nitro-gen removal performances (NH3 → N2) of the nitrogen removal

systems using immobilized cells are summarized in Table 1.The water-jacketed glass column reactor could remove 80 mg ofnitrogen (200 mL of 400 mg L−1ammonia medium) for approxi-mately 100 h [7]. The volumetric nitrogen removal performancewas calculated as 0.61 kg m−3 day−1. The nitrogen removal rateof 5.1 mmol s−1 per cubic meter of the gel (gel load, 25%) wasachieved in a bubble column reactor using double-layer beadswith coimmobilized N. europaea and Pseudomonas sp. [5]. This ratewas equivalent to the volumetric nitrogen removal performance of1.54 kg m−3 day−1. On the other hand, the nitrogen removal systemusing immobilized microorganisms achieved a nitrogen removalrate of 0.066 kg m−3 day−1 [12]. The nitrogen removal systemsusing activated sludge exhibited nitrogen removal performancesof 0.087–0.18 kg m−3 day−1 in the actual thermal power plants.

Our bioreactor containing packed gel envelopes with immobi-lized N. europaea and P. denitrificans yielded a volumetric nitrogenremoval rate of 1.60 kg m−3 day−1 under laboratory conditions [8].In the case of the original envelope, the maximum volumetric nitro-gen removal performance of the system was 0.109 kg m−3 day−1

when nitrate-containing desulfurization wastewater was treated(only denitrification process) [11]. In this study, the volumet-

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bilized cells

obilization Working volume (L) Volumetric nitrogenremoval rate(NH3 → N2) (kg day−1

for 1 m3 reactorvolume)

ilized bylexes

0.0314 0.61

�-carrageenan 0.15 1.54sin 750,000 0.066olymer (PVA-SbQ) 0.25 1.60olymer (PVA-SbQ) 1,800 0.252

ric nitrogen removal performance of the system using modifiedenvelopes with immobilized N. europaea and P. denitrificans was0.252 kg m−3 day−1 in a pilot test. The volumetric performanceachieved using the modified envelopes was twice more than thatachieved using the original envelopes. These differences in the vol-umetric performances using three kinds of envelopes were causedby the gel area of the envelope and working volume. Because thethickness of the envelope was 3 mm and the space between twoenvelopes was 3 mm under laboratory conditions, the gel area of allenvelopes per working volume was 332 m2 m−3. On the other hand,the gel areas of all envelopes—both original and modified—perworking volume were 21.8 and 42.0 m2 m−3 under the pilot-scaleconditions, taking safety into consideration.

At the inflow rate of 0.45 m3 h−1, bacteria immobilized in thegel would propagate and rates of ammonia oxidation and nitro-gen removal increased gradually with time, due to the acclimationunder high nitrogen load in the reactor tank. A little biofilmattached on the surface of gel after nearly two months. Neverthe-less, the bioreactor using thinner packed gel envelopes attainedthe same nitrogen removal performance as that in the laboratoryexperiment without an effect of bacterial attachment, probably dueto the low concentrations of total organic carbon both in the inflowand in the outflow.

5. Conclusion

In this study, the ammonia-containing wastewater from thecoal-fired thermal power plant was continuously treated withthe large-scale bioreactor system. The bioreactor using thinner

packed gel envelopes in which N. europaea and P. denitrificans cellsare coimmobilized could remove nitrogen in the inlet with highremoval efficiency and the total nitrogen concentration in the outletwas maintained at a low level. Furthermore, the maximum utiliza-tion efficiency of the injected electron donor for denitrification wasquite high, and the total organic carbon concentration in the out-let was maintained at a low level. These results indicated that thebioreactor using packed gel envelopes would be highly effectivefor constructing a simple and efficient nitrogen removal systemcapable of simultaneous nitrification and denitrification. In futurestudies, the lamellation of the packed gel envelopes will be essentialin order to increase the volumetric nitrogen removal performance.

Acknowledgment

The authors thank Naho Kitazawa and Haruo Matsumura fortheir help with the analysis and manufacture of the packed gelenvelopes; Ichiro Saitoh, Yoshiyuki Matsuki, Tomohiko Yoshii, Tat-sunori Yoshida, Toshifumi Nishimura, and Takayuki Yanagisawa fortheir valuable advice and discussion.

66 M. Morita et al. / Biochemical Engine

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