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Nitritation performance and biofilm developmentof co- and counter-diffusion biofilm reactors:Modeling and experimental comparison

Rongchang Wanga,b, Akihiko Teradaa, Susanne Lacknera,Barth F. Smetsa,*, Mogens Henzea, Siqing Xiab, Jianfu Zhaob

aDepartment of Environmental Engineering, Technical University of Denmark, Miljoevej, Building 113, DK-2800 Kgs. Lyngby, DenmarkbState Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze Aquatic Environment, Ministry of Education

of China, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China

a r t i c l e i n f o

Article history:

Received 24 September 2008

Received in revised form

6 January 2009

Accepted 4 March 2009

Published online 21 March 2009

Keywords:

Nitritation

Co-diffusion

Counter-diffusion

Biofilm development

Fluorescence in situ hybridization

Membrane-aerated biofilm reactor

* Corresponding author. Tel.: þ45 4525 2230;E-mail address: bfs@env.dtu.dk (B.F. Sme

0043-1354/$ – see front matter ª 2009 Elsevidoi:10.1016/j.watres.2009.03.017

a b s t r a c t

A comparative study was conducted on the start-up performance and biofilm development

in two different biofilm reactors with aim of obtaining partial nitritation. The reactors were

both operated under oxygen limited conditions, but differed in geometry. While substrates

(O2, NH3) co-diffused in one geometry, they counter-diffused in the other. Mathematical

simulations of these two geometries were implemented in two 1-D multispecies biofilm

models using the AQUASIM software. Sensitivity analysis results showed that the oxygen

mass transfer coefficient (Ki) and maximum specific growth rate of ammonia-oxidizing

(AOB) and nitrite-oxidizing bacteria (NOB) were the determinant parameters in nitrogen

conversion simulations. The modeling simulations demonstrated that Ki had stronger

effects on nitrogen conversion at lower (0–10 m d�1) than at the higher values (>10 m d�1).

The experimental results showed that the counter-diffusion biofilms developed faster and

attained a larger maximum biofilm thickness than the co-diffusion biofilms. Under oxygen

limited condition (DO < 0.1 mg L�1) and high pH (8.0–8.3), nitrite accumulation was trig-

gered more significantly in co-diffusion than counter-diffusion biofilms by increasing the

applied ammonia loading from 0.21 to 0.78 g NH4þ-N L�1 d�1. The co- and counter-diffusion

biofilms displayed very different spatial structures and population distributions after

120 days of operation. AOB were dominant throughout the biofilm depth in co-diffusion

biofilms, while the counter-diffusion biofilms presented a stratified structure with an

abundance of AOB and NOB at the base and putative heterotrophs at the surface of the

biofilm, respectively.

ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction suppressed. Advantages such as lower oxygen demands for

Partial nitrification has gained significant recent attention in

wastewater treatment. In partial nitrification, also known as

nitritation, oxidation of nitrite (NO2�) to nitrate (NO3

�) is

fax: þ45 4593 2850.ts).er Ltd. All rights reserved

nitrification and lower organic carbon demands for subse-

quent denitrification are the reason. There are different

approaches to achieve nitritation applicable to suspended

growth or biofilm systems (Pedersen et al., 2003). High

.

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 92700

temperatures and low residence times without biomass

retention favor the growth of aerobic ammonia-oxidizing

bacteria (AOB) over nitrite-oxidizing bacteria (NOB). NOB can

be washed out due to their lower specific growth rate under

these conditions. An example of such a process is the Single

reactor system for High activity Ammonium Removal Over

Nitrite (SHARON) described by Hellinga et al. (2003). Inhibition

of nitrite oxidation can also be controlled by other factors,

such as concentration ratios of NH4þ/NH3, NO2

�/HNO2, in

combination with pH, dissolved oxygen (DO) (Jenicek et al.,

2004), organic carbon concentration (Yun et al., 2004) and

other inhibitors (Peng and Zhu, 2006). Yoo et al. (2006) have

shown that DO control can be more critical than pH control to

attain nitrite build-up in a nitrifying submerged membrane

bioreactor. At DO concentrations of 0.2–0.4 mg L�1, about 70%

ammonium conversion to nitrite was obtained with total

ammonium removal higher than 93% (Yoo et al., 2006).

However, several questions still remain. In particular it has

been shown that NOB can adapt to initially inhibiting condi-

tions, which compromises continued nitrite production

(Antonisen et al., 1976; Abeling and Seyfried, 1992).

Membrane aerated biofilm reactors (MABR) have been

introduced to remove nutrients (nitrogen or phosphorus or

both) (Semmens et al., 2003; Terada et al., 2003, 2006; Downing

and Nerenberg, 2008) or other pollutants (Ohandja and

Stuckey, 2007) from wastewaters. In MABR systems, the

oxygen is supplied to the base of the biofilm through a gas-

permeable membrane and the substrate is supplied to the

surface of the biofilm from the bulk liquid; we have termed the

resulting system a counter-diffusion biofilm. One advantage of

MABRs is the possibility for 100% oxygen transfer efficiency

(Brindle et al., 1998), which is not achievable with conventional

biofilm systems, which we have termed co-diffusion biofilms.

Further advantages of membrane-aerated systems are

minimal stripping of volatile organic compounds, controlled

delivery of potentially explosive gases, and a well-defined

surface area for mass transfer (Shanahan and Semmens,

2006). The independent control of oxygen surface loading (LO2)

and ammonia nitrogen surface loading (LNH4) makes the MABR

theoretically more amenable for successful nitritation.

Implementation of nitrogen removal via the nitrite pathway

has been obtained in sequencing batch processes (e.g., Pam-

brun et al., 2006), but few have been achieved with continuous

flow processes (Fux et al., 2004; Peng and Zhu, 2006). However,

effective control and manipulation of co- and counter-diffu-

sion biofilm geometries may provide some new clues to ach-

ieve nitritation in continuous-flow biofilm reactors.

Understanding the reaction and diffusion processes in

membrane aerated biofilms is necessary for optimizing the

operation conditions of a MABR. The mass transfer coefficient

for oxygen (Ki) seems to be a crucial parameter in these reac-

tors. Also the maximum biofilm thickness (Lf,max) has

a significant influence on the performance of membrane-

aerated biofilms.

The purpose of this study was therefore to (1) examine the

effects of Ki and Lf,max on the nitrogen conversion perfor-

mance of co- and counter-diffusion biofilm reactors; (2) vali-

date the feasibility of implementing and controlling partial

nitrification in continuous-flow co- and counter-diffusion

biofilm reactors; and (3) compare nitritation performance of

co- and counter-diffusion biofilms during their development

through modeling and experimental investigation.

2. Materials and methods

2.1. Reactor configuration

Two structurally identical reactors (R1 and R2) were con-

structed to support co- and counter-diffusion biofilms,

respectively. Fig. 1 shows the configuration of both systems

schematically. Briefly, each reactor had a working volume of

0.59 L and was completely mixed via liquid recirculation (the

average recirculation flow rate was approx 42.9 L h�1, upflow

velocity approx. 8.5 m h�1). Residence time distribution (RTD)

tests revealed dimensionless dispersion numbers of 0.37 and

0.38 for both reactors, respectively, indicating completely

mixed conditions. Gas-permeable silicone (J. Lindemann

GmbH, Helmstedt, Germany; 250 mm thickness, 12 mm diam-

eter, 300 mm length) was used as gas delivery membrane and

support for biofilm growth. The co-diffusion system (R1) had

a hydrophobic, microporous polyethylene hollow-fiber bundle

(128 fibers) in the recirculation line (MHF3504; Mitsubishi

Rayon Co. Ltd., Tokyo, Japan) to deliver oxygen to the bulk

liquid. DO concentration in the bulk was kept below

0.5 mg L�1. Therefore, both oxygen and ammonium were

supplied to the biofilm in the main reactor compartment from

the liquid phase in the co-diffusion geometry system (R1). In

R1, the gas lumen of the silicone membrane was filled with

nitrogen gas and sealed, preventing oxygen flux from the

lumen side. In the counter-diffusion system (R2), oxygen was

supplied directly from the silicone membrane to the biofilm

whereas ammonium was provided from the liquid phase.

Pressurized filtered air was used as oxygen source. The air

pressure before entering the hollow fibers or silicone

membrane was controlled at 1.8 � 0.2 kPa above atmospheric

pressure. To minimize biofilm growth on surfaces other than

the silicone membrane, the reactor walls and the hollow fiber

membrane module in the co-diffusion system (R1) were

scoured and cleaned twice a week with ultrasonic and

mechanical methods.

2.2. Medium

Both biofilm reactors were fed with the same synthetic

medium using peristaltic pumps (Ismatech MV-MS/CA8C,

Glattburg, Zurich, Switzerland). The feed was prepared with

tap water and contained NH4HCO3 (N source), NaHCO3 (C

source, pH buffer), 0.03 g phosphorus (as K2HPO4) L�1 and

1 ml L�1of a trace element solution (after Pynaert et al., 2004).

The pH was adjusted to 8.0–8.3. The influent ammonium

concentration and flow rate were adjusted during the exper-

iment to obtain the desired N load; the influent NaHCO3

concentration varied depending on the required buffer

capacity and pH.

2.3. Inoculation and start-up

Both systems were inoculated with activated sludge from

a lab-scale nitrifying reactor which displayed full and stable

c d

Silicone tube

Air

Influent Nutrient medium

Recycling pump

Hollow Fiber Aeration ModuleEffluent

pH/ORP meterDO meter

PAir

Silicone tube

Influent Nutrient medium

Recycling pump

Effluent

pH/ORP meterDO meter

a b

Fig. 1 – Schematic of metabolite fluxes in the reactors housing the co- (a and c) and counter-diffusion biofilms (b and d).

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 9 2701

nitrification at an influent NH4þ-N concentration of 250 g-

N m�3. After inoculation, both systems were operated in batch

mode until ammonia removal was detected. Then the opera-

tion was switched to continuous mode. The first day of the

continuous mode operation was recorded as the first day of

operation time (day 1).

2.4. Chemical analysis

The concentrations of NH4þ-N, NO2

�-N, and NO3�-N were

measured with Spectroquant� test kits (Merck KGaA,

Germany). DO was measured online with a DO electrode (YSI

Model 5331, Yellow Springs, OH, USA) and continuously

acquired on a personal computer connected to the DO

monitor (YSI Model 5300, Yellow Springs, OH, USA) by a multi-

channel data acquisition device (LabPCþ, National Instru-

ments, Austin, TX, USA). N2O measurements were carried out

off-line with a 1321 Photoacoustic Multi-gas Monitor (Innova

AirTech Instruments A/S, Denmark).

2.5. Biofilm thickness measurements

To obtain periodic biofilm samples, the silicone membrane

was pushed upwards after shutting off all pumps, and a 0.5 cm

terminal end was cut-off with a sharp blade. The entire

sample was immediately fixed in 4% paraformaldehyde

solution (pH 7.2) for 3 h. After fixation, biofilm samples were

submerged overnight in Tissue-Tek� O.C.T.� Compound

(Sakura Finetek Europe B.V., the Netherlands). The sample

was frozen at �25 �C and sectioned into 10 mm-thick vertical

slices with a cryomicrotome (Reichert-Jung Cryocut 1800,

Leica). The produced slices were mounted on microscope

slides. Washing and dehydration were performed according to

reported procedures (Schramm et al., 1996). Mounted biofilm

samples were stained with Syto9 (Molecular Probes Inc., USA)

and then observed under a confocal scanning laser micro-

scope (CLSM) (Leica TSC SP5, Germany). Biofilm thickness was

measured directly with the CLSM and the average biofilm

thickness was calculated with at least 50 positions for each

sample. A shrinking factor (a) of 1.89 was calculated by

comparing the biofilm thickness before and after the fixation

procedure. The shrinking factor was used to convert biofilm

thickness based on staining and CLSM observation to actual

biofilm thickness.

2.6. Fluorescence in situ hybridization (FISH)

To investigate the spatial distribution of AOB and NOB in

biofilms, FISH was performed according to established proto-

cols (Amann et al., 1995). The used oligonucleotide probes and

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 92702

their fluorochromes are shown in Table 2. The final probe

concentration was approximately 5 ng mL�1. The formamide

concentration (FA) in the hybridization buffer was 35%.

Hybridization was carried out at 46 �C for 3 h. Subsequently,

a stringent washing step of the samples was performed at

48 �C for 15 min in 50 ml of prewarmed washing solution

containing sodium chloride, Tris–HCl and EDTA. Micrographs

of the hybridized slices were obtained with a CLSM observa-

tion with argon (488 nm) and HeNe lasers (543 and 633 nm)

illumination.

2.7. Model development and simulation

2.7.1. Model developmentA one-dimensional multi-population biofilm model was con-

structed in AQUASIM 2.1 (Reichert, 1998). Both co- and

counter-diffusion biofilm geometries were modeled to simu-

late performance and microbial community structure under

various operational conditions. The models have the same

structural dimensions and membrane surface areas as in the

experimental biofilm reactors. Both modeled biofilm reactors

have two linked compartments: a completely-mixed gas

compartment and a biofilm compartment, the latter of which

is further composed of a biofilm matrix and bulk liquid. The

gas compartment, with oxygen partial pressure of 21 kPa, is

connected to the base of the biofilm for the counter-diffusion

system and to the bulk liquid for the co-diffusion system,

respectively. The oxygen flux (JO2) from the gas compartment

(the silicone tube for the counter-diffusion geometry or the

hollow fiber membrane module for the co-diffusion geometry)

to the biofilm base or the bulk liquid is given by equation (1):

JO2 ¼ Ki

�SO2;g

H� SO2;bb

�or JO2 ¼ Ki

�SO2;g

H� SO2;BL

�(1)

where SO2,g, SO2,bb and SO2,BL are the oxygen concentrations in

the gas compartment and at the biofilm base or in the bulk

liquid, respectively (g-O2 m�3), Ki is the oxygen mass transfer

coefficient (m d�1), H is the non-dimensional Henry’s law

coefficient. Gas transfer of nitrogen gas and carbon dioxide

were not considered in this model.

For all simulations, the initial biofilm thickness was set as

10 mm and the initial biomass was assumed to contain 50%

AOB, 25% NOB and 25% heterotrophic bacteria (HB). Biofilm

detachment was modeled by employing a pseudo steady-state

approach using the following expression: udet ¼ uF $

(Lf /Lf,max)2 when uF > 0 (otherwise udet ¼ 0), with udet: biofilm

detachment rate, Lf,max: the maximum biofilm thickness and

uF: the biofilm growth velocity (Lackner et al., 2008).

2.7.2. Sensitivity analysisA normalized absolute–relative sensitivity function was used

to investigate the identifiability and relative significance of

each parameter on reactor performance. It is defined as

(Reichert, 1998):

dCi ;p ¼ pvCiðpÞ

vp

�g�N m�3

�(2)

where dCi ;p, Ci, and p are the absolute–relative sensitivity

function, the effluent concentration of the considered species

(NH4þ-N, NO2

�-N, or NO3�-N), and the considered model

parameter, respectively. The dCi ;p measures the relative

change in Ci for a 100% change in p and all these changes are

calculated in linear approximation. The analysis was per-

formed over the same time interval as the typical simulation.

2.7.3. Model simulation and calibrationActivated Sludge Model No. 1 was extended by implementing

a two-step nitrification process (Henze et al., 2000) (Table I in

Appendix A). Bacterial growth and decay processes were

considered for HB (XH), AOB (XAOB) and NOB (XNOB) (Table II in

Appendix A). Oxygen (SO) was used as an electron acceptor for

both autotrophic and heterotrophic growth, while denitrifying

HB also use nitrite (SNO2) and nitrate (SNO3) as electron

acceptors when oxygen becomes limiting. Kinetic and stoi-

chiometric parameters for HB were derived from literature

(Table III in Appendix A). A wide range of maximum specific

growth rates for AOB (mmax,AOB) and NOB (mmax,NOB) has

reported in the literature. To increase the accuracy of the

simulation, the values of mmax,AOB and mmax,NOB were

confirmed by parameter estimation based on in situ respiro-

metric experiments (results not shown) in co- and counter-

diffusion biofilms, respectively (Table III in Appendix A).

3. Results and discussion

3.1. Sensitivity analysis

A summary of the sensitivity analysis of key parameters on

the NH4þ-N, NO2

�-N, and NO3�-N concentrations in the effluent

of co- and counter-diffusion biofilm systems is provided in

(Table V Appendix A). The most crucial parameter for

controlling nitritation performance in counter-diffusion bio-

film reactors was Ki, while mmax,AOB was the most determinant

parameter in the co-diffusion biofilm reactors. Ki had a less

significant effect on the nitritation performance in the co-

diffusion biofilm system. As expected, mmax,AOB and mmax,NOB

had strong effects on the nitrite accumulation in both co- and

counter-diffusion biofilm systems, especially in the co-diffu-

sion biofilm system. The biokinetic and stoichiometric

parameters related to HB were of minor significance. These

results indicate that it is important to control the oxygen flux

and keep AOB as the key player in biofilm systems in order to

maintain a high nitritation performance, irrespective of bio-

film geometry.

3.2. Effects of Ki and Lf,max on nitritation performance

The effects of Ki and Lf,max on the nitrogen conversion

performance in co- and counter-diffusion biofilms are shown

in Fig 2. In both co- and counter-diffusion biofilm reactors, the

effluent NH4þ-N concentration decreases with Ki increases,

which indicates that a higher oxygen mass transfer coefficient

enhances the oxidation of ammonia as more oxygen is

available to AOB in both systems (Fig. 2a and b). The effluent

NH4þ-N concentration increases with Lf,max in both co- and

counter-diffusion biofilm systems. The reason for the small

peak at Lf,max 100 mm in the co-diffusion scenario at high Ki

values is not clear. Fig. 2a and b indicate that satisfactory

a b

c d

ef

145150155160165170175180185

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400600

800

05

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20

Efflu

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t N

H4

+-N

co

nc. [g

N/m

3]

Ki [m

/d]

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05

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20

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400600

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05

1015

20

Efflu

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t N

O3

--N

co

nc. [g

N/m

3]

L_f_max [ m]

Ki [m

/d]

051015202530

130140150160170180190

200

0500100015002000

2500

010

2030

40L_f_max [ m]

L_f_max [ m]

L_f_max [ m]

L_f_max [ m

]

130140150160170180190200

0102030405060

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5001000

15002000

25003000 0

1020

3040

Efflu

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t N

O3

--N

co

nc. [g

N/m

3]

Ki [m

/d]

010203040506070

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0500100015002000

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/d]

Ki [m

/d]

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O2

--N

co

nc. [g

N/m

3]

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/d]

101520253035404550

L_f_max [ m]

Fig. 2 – Predicted steady-state effluent nitrogen concentrations in co- (a, c, e) and counter- (b, d, f) diffusion biofilm reactors at

different values of Ki and Lf,max after 120 days. (a, b) NH4D-N; (c, d) NO2

L-N; (e, f) NO3L-N.

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 9 2703

ammonia removal efficiency is obtained if biofilm thickness

remain below 500 mm. In addition, ammonia removal effi-

ciency increases dramatically when Ki increases from 0.5 to

10 m d�1, but additional Ki increases give marginal improve-

ments, especially at biofilm thickness larger than 800 mm.

An increase in Ki has a completely different effect on nitrite

production in co- versus counter-diffusion biofilm systems

(Fig. 2c and d). In counter-diffusion systems, the nitrite

concentration in the bulk liquid decreases as the Ki increases,

especially from 0.5 to 10 m d�1. On the contrary, the bulk

liquid nitrite concentration in the co-diffusion biofilm system

increases when Ki increases. Clearly, an increase of Ki

compromises the nitrite accumulation in the counter-diffu-

sion biofilm system but enhances the nitrite accumulation in

the co-diffusion biofilm system investigated in this study.

There is a small valley in nitrite concentrations at Lf,max values

near 200 mm in counter-diffusion biofilms and at about 100 mm

in the co-diffusion biofilms. When Lf,max increases even

further (larger than 800 mm in counter-diffusion biofilms and

larger than 400 mm in co-diffusion biofilms), Lf,max appears to

have no significant effect on the nitrite production (Fig. 2c

and d).

Nitrate production is improved dramatically when Ki

increases from 0.5 to 20 m d�1 and then levels off for both co-

Table 1 – Target operational conditions for the co- and counter-diffusion biofilm reactors.

Strategy Time (days) pH DO (mg L�1) NH4þ-N loading (g-N L�1 d�1)

Onset 0–14 6.5–7.5 1.0–5.0 0.21–0.26

Low DO 15–40 7.2–7.8 0.1–0.5 0.21–0.26

Low DO þ High pH 40–52 7.5–8.0 0.05–0.1 0.21–0.26

Low DO þ High pH þ High NH4þ-N load 53–140 8.0–8.3 0.04–0.05 0.63–0.78

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 92704

and counter-diffusion biofilms. The effects of Lf,max on the

nitrate production are opposite to those of nitrite production,

i.e., a small peak is observed at Lf,max values of 200 and 100 mm

in counter- and co- diffusion biofilms; and the effect of Lf,max

becomes marginal above biofilm thicknesses of 800 mm and

400 mm in counter- and co- diffusion biofilms, respectively.

From Fig. 2c–f, it can be concluded that at biofilm thickness

exceeding 800 and 400 mm in counter- and co-diffusion bio-

films, further increases have no significant effects on either

nitrite or nitrate production, suggesting an optimum range of

Lf,max for nitritation performance in both co- and counter-

diffusion biofilm systems.

3.3. Comparison of nitritation performance in co- andcounter-diffusion systems

The DO concentration in the bulk liquid (SO2,BL) of the reactors

was controlled by adjusting the air pressure in the membrane

lumen (Fig. I in Appendix A). The variations of SO2,BL are

summarized in Table 1. During the first 5 days, SO2,BL was

about 2.0–5.0 mg L�1 at a relatively low air pressure (approx.

2.0 kPa). Oxygen consumption in both co- and counter-diffu-

sion systems was relatively low because of the low biomass

concentration in the reactors. When biofilms developed on the

membrane surface, SO2,BL decreased dramatically to below

1.0 mg L�1 at the same applied pressure. Then the air pressure

was increased up to 6 kPa to supply enough oxygen for biofilm

formation for several days. Formation of bacterial colonies

and thin biofilms (about 10 mm) on the membrane surface was

confirmed with microscopic observation. From day 15 onward

the air pressure was maintained at approx. 1.8 kPa for both

reactors.

Table 2 – Oligonucleotide probes used for fluorescence in situ

Probe Names in oligonucleotideprobe database (OPD)

Sequenc

EUB338 S-D-Bact-0338-a-A-18 GCT GCC TCC CG

EUB338II S-*-BactP-0338-a-A-18 GCA GCC ACC CG

EUB338III S-*-BactV-0338-a-A-18 GCT GCC ACC CG

non EUB S-*-Bact-338-a-B-18 ACT CCT ACG GG

Nso190 S-F-bAOB-0189-a-A-19 CGA TCC CCT GC

Nso1225 S-F-bAOB-1224-a-A-20 CGC CAT TGT AT

Nmo218 – CGG CCG CTC CA

Nsm156 S-G-Nsm-0155-a-A-19 TAT TAG CAC AT

Cluster6a192 – CTT TCG ATC CC

Cluster6a192 comp – CTT TCG ATC CC

NIT3 S-G-Nbac-1035-a-A-18 CCT GTG CTC CA

CNIT3 – CCT GTG CTC CA

Ntspa662 S-G-Ntspa-662-a-A-18 GGA ATT CCG CG

cNtspa662 – GGA ATT CCG CT

Nitrogen conversion in both systems is shown in Fig. 3.

During the first 10 days, the ammonia removal of R1 and R2

increased steadily from 8.5% to 80.1% and from 15.5% to 82.4%,

respectively. SO2,BL was reduced from above 4.0 mg L�1 to below

1.0 mg L�1. The ammonia removal decreased from 91.0% (day

11) to 45.7% (day 15) for the co-diffusion and from 82.4% (day 10)

to 48.2% (day 13) for the counter-diffusion system. However,

the ammonia removal performance of both systems recovered

(Fig. 3a,b), reaching 99.7% on day 31 for the co-diffusion system

and 99.1% on day 21 for the counter-diffusion system.

In order to stimulate and maintain partial nitrification in

both systems, the influent ammonium-nitrogen concentration

was increased from 67 mg L�1 to 262 mg L�1 from day 53 to day

60 (corresponding to an increase in NH4þ-N loading from 0.21 g-

NH4þ-N L�1d�1 to 0.78 g-NH4

þ-N L�1d�1). Nitrite accumulation

was observed in both systems, but the trends were very

different. In the counter-diffusion system, the nitrite concen-

tration increased slowly from about 3.2 mg-NO2�-N L�1 (day 60)

to 20.5 mg-NO2�-N L�1 (day 100), then it stayed at about 20 mg-

NO2�-N L�1 until the biofilm sloughing event on day 140, the last

day of the reactor operation, when almost all biofilm detached

from the membrane. The NO3� concentration in this system

was relatively constant and fluctuated around 45 mg-NO3�-

N L�1. The NO2� concentration in the co-diffusion system

increased gradually from 4.6 mg-NO2�-N L�1 (day 60) to about

70 mg-NO2�-N L�1 (day 100) where it was maintained

throughout the operational period. The NO3� concentration in

the co-diffusion system continually decreased from 56.2 mg-

NO3�-N L�1 to 26.4 mg-NO3

�-N L�1. In both systems, the effluent

NH4þ-N concentration increased after the load increase from

about 11 mg-NH4þ-N L�1 (before day 60) to about 140 mg-NH4

þ-

N L�1 (from day 60 to day 80); it decreased gradually to about

hybridization (FISH).

e (50–30) Target organism(s) Dye

T AGG AGT Domain Bacteria FLUO

T AGG TGT Planctomycetales FLUO

T AGG TGT Verrucomicrobia FLUO

A GGC AGC None FLUO

T TTT CTC C b-Subgroup ammonia-oxidizing bacteria Cy3

T ACG TGT GA b-Subgroup ammonia-oxidizing bacteria Cy3

A AAG CAT Nitrosomonas oligotropha Cy3

C TTT CGA T Nitrosomonas spp., Nitrosococcus mobilis Cy3

C TAC TTT CC N. oligotropha lineage (cluster 6a) Cy3

C TGC TTC C Competitor for cluster 6a192 Cy3

T GCT CCG Nitrobacter Cy5

G GCT CCG Competitor for NIT3 Cy5

C TCC TCT Nitrospira Cy5

C TCC TCT Competitor for Ntspa662 Cy5

NO2--N,

127.4 mgN/d,30.0%

NH4+-N,

177.0 mgN/d,41.7%

NO3--N,

46.0 mgN/d,10.8%

N2O,3.7 mgN/d,

0.9%

Unidentified,70.6 mgN/d,

16.6%a

NO2--N,

37.2 mgN/d,8.8%

NH4+-N,

122.1 mgN/d,28.8%Unidentified,

160.0 mgN/d,37.7%

N2O,1.9 mgN/d,

0.5%NO3

--N,103.5 mgN/d,

24.4%

b

Fig. 4 – Nitrogen mass balance (mg-N dL1) in the co- (a) and

counter- (b) diffusion biofilm reactors at 120 days with

influent total N of 424.8 mg-N dL1.

a

Time (d)

0 20 40 60 80 100 120 140

0 20 40 60 80 100 120 140

N co

ncen

tratio

n (m

g/L

)

0

50

100

150

200

250

300

NH4_inf.NO3_effNO2_effNH4_effNO3_simulatedNO2_simulatedNH4_simulated

b

Time (d)

N co

ncen

tratio

n (m

g/L

)

0

50

100

150

200

250

300

NH4_inf.NO3_effNO2_effNH4_effNO3_simulatedNO2_simulatedNH4_simulated

Fig. 3 – Comparison of simulated and actual performance

in co- (a) and counter- (b) diffusion biofilm reactors.

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 9 2705

90–100 mg-NH4þ-N L�1 (from day 80 to day 140) indicating a slow

increase in ammonia removal capacity in both systems.

Comparisons of the experimental and the model simulated

effluent concentrations in both systems are also shown in

Fig. 3. The simulated nitrite effluent profiles of both systems

roughly match the trend in the experimental profiles. There is,

however, a significant deviation between the predicted and

observed NH4þ effluent concentration, especially after day 80.

These differences may be due to an underestimation of either

the heterotrophic denitrification or anaerobic ammonium

oxidation activity in the simulation, but it is also possible that

differences in the oxygen flux caused this deviation. In addi-

tion, in the co-diffusion system, the nitrate production was

underestimated after the increase of NH4þ loading rate on day

53. The model predicts a rapid decrease in nitrate and increase

in nitrite after increasing the NH4þ loading, unlike the experi-

mental trends. The AOB and NOB biokinetic parameters in the

model, obviously, overpredict the NOB disappearance more

than the experiment. NOB may be more persistent than

previously assumed: the persistence of Nitrobacter NOBs in

bioreactor even under oxygen limited conditions has recently

been shown by others (Ahn et al., 2008).

The percentage of each nitrogenous component in the

effluent on day 120 is shown in Fig. 4. The ammonia loading

rate at 120 days was 424.8 mg-N d�1 for both systems. The

removed ammonia was 247.8 mg-N d�1 (58.3%) and 302.7 mg-

N d�1 (71.3%) in co- and counter-diffusion system, respec-

tively. Although the ammonia removal rate is lower in the co-

diffusion system, a higher nitrite production rate was ach-

ieved (127.4 mg-N d�1) compared to the counter-diffusion

system (37.2 mg-N d�1). The poorer nitrite build-up in the

counter-diffusion system is probably caused by its inherent

biofilm geometry: oxygen and produced nitrite are the highest

at the same location, allowing NOB to grow at the biofilm base.

A recent numerical study, using a two-dimensional cellular

automaton (CA)-hybrid model, confirmed that NOB can

compete more effectively for oxygen and substrate in counter-

versus co-diffusion biofilm system (Bell et al., 2005).

The N2O concentration varied from 50.2 to 135.8 ppm in the

co-diffusion system (air flow rate in membrane lumen

Qair ¼ 22.6 L d�1) and from 3.1 to 7.5 ppm in the counter-diffu-

sion system (Qair ¼ 210.4 L d�1). The N2O concentrations at

120 day were 129.7 � 7.1 ppm and 7.2 � 0.2 ppm translating in

140

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 92706

N2O production rates of 3.7 � 0.2 mg-N d�1 and 1.9 � 0.1 mg-

N d�1, for the co- and counter-diffusion systems respectively.

Thus, only a very small part of removed N (less than 1%) was

due to N2O production in both systems. The rest of the

unidentified N was about 16.6% and 37.7% in the co- and

counter-diffusion systems, respectively. The nitrogen fluxes

due to free ammonia stripping from the bulk liquid to the

membrane lumen in both systems were calculated to be less

than 0.1% of the total nitrogen loading. Based on these mass

balances, it can be speculated that either denitrification

(heterotrophic or autotrophic) or anaerobic ammonium

oxidation occurred in both systems and most of the unidenti-

fied N loss could be in the form of N2 gas. An insignificant

degree of heterotrophic denitrification was predicted

(<1 mg L�1, results not shown) because of small organic carbon

fluxes driven by nitrifying bacterial decay or their extracellular

polymeric substances. Nitrogen losses from autotrophic bio-

films have previously been reported as caused by anaerobic

ammonia oxidation (Kuai and Verstraete, 1998; Helmer et al.,

2001). Another possible sink could be autotrophic denitrifica-

tion by AOB as previously reported (Kampschreur et al., 2006).

However, more work is needed to identify the responsible

mechanisms to close the nitrogen balance in these systems.

3.4. Biofilm development

Comparison of the experimentally measured and the model

predicted values biofilm thickness are shown in Fig. 5. The

biofilm thickness measured on day 140 was 107 � 31 mm for

the co-diffusion system and 887 � 161 mm for the counter-

diffusion system. Based on these results, the Lf,max values

used in this study for the co- and counter-diffusion biofilm

systems are set to 150 mm and 1000 mm, respectively.

The counter-diffusion system had a higher biofilm growth

rate and a higher Lf,max than the co-diffusion biofilm. The

increase in NH4þ concentration (from day 53 to day 60) had

a small effect on the biofilm thickness in the co-diffusion

system versus the counter-diffusion system.

Time (d)

0 20 40 60 80 100 120 140 160

Bio

film

th

ic

kn

es

s ( m

)

0

200

400

600

800

1000

1200

Co-diffCounter-diff Co-diff simulatedCounter-diff simulated

Fig. 5 – Biofilm thickness profiles vs. time in co- and

counter-diffusion biofilms.

Biofilm thickness affects the substrate mass transfer

processes in the biofilm and through the membrane, which are

both vital to the reactor performance in the membrane-assis-

ted biofilm systems (Celmer et al., 2008). In addition, bacterial

growth and respiration near the membrane surface potentially

enhances oxygen transfer across the membrane wall as shown

before (Freitas dos Santos and Livingston, 1995; Semmens and

Essila, 2001). When biofilm thickness on the membrane

substratum increases, redox-stratification may result, which

makes a biofilm feasible for complete nitrogen removal via

either heterotrophic (e.g., Semmens et al., 2003) or autotrophic

pathways (e.g., Terada et al., 2007; Lackner et al., 2008). In this

study, the DO concentration in the bulk liquid was almost

undetectable, normally lower than 0.05 mg-O2 L�1 after day 60

at which point the biofilm thickness was higher than 200 mm in

counter-diffusion biofilm system. As the biofilm thickness on

day 140 was 887 � 161 mm in the counter-diffusion system,

redox-stratification was likely established in these biofilms.

On the contrary, the biofilm thickness reached only 150 mm in

the co-diffusion system. This might contribute to the differ-

ence in terms of nitrogen loss as shown in Fig. 4 (37.7% in

counter-diffusion biofilm vs. 16.6% in co-diffusion biofilm).

The relationships between biofilm thickness and nitrite

production rate in co- and counter-diffusion systems are

shown in Fig. 6. The solid dots represent the situation in the co-

diffusion system. Although the maximum biofilm thickness

was less than 150 mm, the nitrite production rate increases

dramatically from 46.0� 0.4 mg-N d�1 to 125.7 � 1.8 mg-N d�1

when the biofilm thickness develops from 66.5� 21.8 mm to

107.7 � 30.6 mm. However, in counter-diffusion biofilms, even

at biofilm thickness as large as 887 � 161 mm, the nitrite

production rate is still below 40 mg-N d�1. Effective control of

biofilm thickness is critical for optimizing the nitritation

performance of biofilm reactors, especially for counter-diffu-

sion biofilm geometries. The significance of biofilm thickness

control has been explored numerically (Terada et al., 2007) or

experimentally (Semmens et al., 2003; Celmer et al., 2008) for

0 200 400 600 800 1000

Nitrite p

ro

du

ctio

n rate (m

gN

/d

)

0

20

40

60

80

100

120

Co-diffusionCounter-diffusion

Biofilm thickness ( m)

Fig. 6 – Relations between biofilm thickness and nitrite

production rate in co- and counter-diffusion systems (C,

co-diffusion system; B, counter-diffusion system).

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 9 2707

membrane-aerated and supported biofilms. Optimal biofilm

thickness control strategies need to be further investigated so

as to maintain stable and high nitrite accumulation.

3.5. Comparison of microbial community structure andspatial distribution

The spatial distribution of AOB and NOB in both co- and

counter-diffusion biofilms was investigated by FISH. Repre-

sentative FISH images for co- and counter-diffusion biofilms

are shown in Fig. 7. AOB in the biofilm sections hybridize with

both EUB mix (green) and Nso series (red), so they are yellow in

the two-channel overlapped confocal images. Analogously,

NOB hybridize with both EUB mix (green) and NIT3 or

Ntspa662 (blue), and they are blue to cyan in Fig. 7.

The biofilm samples were harvested on days 40 and 120 of

operation. The bold white lines show the bases of biofilms (i.e.,

the positions of silicone membrane) on the sections. On day 40

Fig. 7 – Confocal laser scanning micrographs of biofilm sections

EUB338mix-FLUO (green) targeting most Bacteria; (Nso190-Cy3 D

and NIT3-Cy5 (blue) specific for Nitrobacter spp. Sections were tak

(b, d) diffusion biofilm reactors.

the co-diffusion biofilm is looser and more protuberant, while

the counter-diffusion biofilm is more compact and tight

(Fig. 7a and b). The b-subgroup AOB were dominant in all the

observed biofilm samples and NOB belonging to the genus

Nitrobacter were located in the regions adjacent to the silicone

membrane surface or biofilm surface. The probe Ntspa662 was

used to check the presence of Nitrospira signals in the biofilm;

however, Nitrospira spp. were not detected in both systems.

A different spatial distribution was observed on day 120

(Fig. 7c and d). First, the thickness of the counter-diffusion

biofilm was much higher than that of the co-diffusion biofilm.

Second, the counter-diffusion biofilm had a clear stratified

structure: most of AOB and NOB were located near the base of

the biofilm, i.e., close to the surface of the membrane as the

supply source of oxygen, and nonAOB nonNOB (presumable

HB) were located at the surface of the biofilm close to the bulk

liquid (Fig. 7d). A similarly layered biofilm structure has been

reported before (Hibiya et al., 2003; Lapara et al., 2006).

. All sections were simultaneously hybridized with

Nso1225-Cy3 D Nmo218-Cy3) (red) targeting most AOB,

en at 40 (a, b) and 120 (c, d) days from co- (a, c) and counter-

w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 6 9 9 – 2 7 0 92708

On the contrary, such stratified structure was not observed

in the co-diffusion biofilm (Fig. 7c), where AOB were dominant

and the few NOB were located at the biofilm base. Compared

to the counter-diffusion biofilm, the co-diffusion biofilm was

thinner, which probably resulted in deeper penetration of

ammonia which, in turn, may have inhibited NOB, allowing

a much higher nitrite accumulation and lower nitrate

production in the co-diffusion biofilm systems. Lydmark et al.

(2006) have similarly observed more NOB at the base than the

top of nitrifying biofilms in a trickling filter.

4. Conclusions

Co- and counter-diffusion biofilm reactors were operated

under oxygen limited conditions in order to initiate and

maintain stable nitritation processes. The experimental

results show that a combination of high pH (8.0–8.3) and high

ammonia loading rates (about 0.78 g-NH4þ-N L�1 d�1) were

necessary to trigger NOB inhibition and obtain nitrite accu-

mulation. Significant nitritation was achieved with co-diffu-

sion biofilms but very poor nitritation was obtained with

counter- diffusion biofilm systems. Sensitivity analysis

revealed that the oxygen mass transfer coefficient (Ki) and

maximum biofilm thickness (Lf,max) were most determinant in

the nitrogen conversion performance for both biofilm

systems. The biofilms obtained in the counter-diffusion

systems were much thicker than those obtained in the co-

diffusion systems. In addition, NOB survived to a much

greater extent in the counter- versus the co-diffusion biofilm

after several months of operation. Our study reveals the

difficulty in obtaining partial nitrification in biofilm-based

nitrifying reactors, which is exacerbated in membrane-

aerated and membrane supported biofilms, where the

inherent system geometry may complicate inhibition and

washout of NOB, a condition necessary to ensure sufficient

and stable nitritation.

Acknowledgments

This research was supported by the Sino-Danish Scientific

Cooperation Program (S14NNP28) and the DANIDA fellowship,

the Danish Agency for Science Technology and Innovation

(FTP-ReSCoBiR), Program for Young Excellent Talents in

Tongji University (2007KJ015). We are also grateful to Dr.

Laurent Lardon, Ms. Lene K. Jensen, Dr. Najoi El Azhari, Mr.

Sanin Musovic, Mr. Anders Fredenslund and Mr. Xiangwen

Kong for assistance in various aspects of the work.

Appendix A.Supplemental material

Supplementary information for this manuscript can be

downloaded at doi: 10.1016/j.watres.2009.03.017.

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