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Improving nitrogen removal in an ANAMMOXreactor using a permeable reactive biobarrier

Fangang Meng a,b,*, Guangyi Su a,b, Yifang Hu a,b, Hui Lu a,b,Li-Nan Huang c, Guang-Hao Chen a,d

aSYSU-HKUST Research Center for Innovative Environmental Technology (SHRCIET), School of Environmental

Science and Engineering, Sun Yat-sen University, Guangzhou 510275, PR ChinabGuangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou

510275, ChinacSchool of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR ChinadDepartment of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Clear

Water Bay, Hong Kong, China

a r t i c l e i n f o

Article history:

Received 13 November 2013

Received in revised form

16 March 2014

Accepted 18 March 2014

Available online 27 March 2014

Keywords:

ANAMMOX

Biofilm reactors

Mass transport

Membrane fouling

Nitrogen removal

* Corresponding author. SYSU-HKUST ReseaScience and Engineering, Sun Yat-sen Unive

E-mail address: mengfg@mail.sysu.edu.cn

http://dx.doi.org/10.1016/j.watres.2014.03.0490043-1354/ª 2014 Elsevier Ltd. All rights rese

a b s t r a c t

A novel ANAMMOX biofilm reactor that combines the advantages of conventional biofilm

reactors and membrane bioreactors (MBRs) was developed in an attempt to decrease the

levels of nitrogen in the reactor filtrate. In this reactor, nonwoven fabric modules served as

both biofilm carriers and membrane-like separators, and the biofilm acted as a permeable

reactive barrier for the removal of nitrogen species from the bulk liquid. Long-term

monitoring suggests that the nitrogen removal rates (NRR) of the reactor reached ca.

1.6 kg-N/(m3 d). Interestingly, large fractions of the ammonium (ca. 27%) and nitrite (ca.

48%) remaining in the bulk liquid were removed during their transport through the biofilm;

thus, the reactive barrier process of the biofilm contributed ca. 11% to the total NRR. With

an increase in the imposed flux, the contribution of the reactive barrier process to the

removal of nitrogen from the reactor bulk liquid increased significantly, e.g., it contributed

26% to the NRR at 17.4 L/(m2 h). Additionally, the nonwoven modules could retain free

bacteria effectively; they maintained a non-fouling state during the entire operation period

of approximately 400 days. Sequence analysis shows that Candidatus Kuenenia-like species

dominated the ANAMMOX bacteria in the reactor. These results clearly demonstrate that

this innovative reactor holds great promise for improving the ANAMMOX process, thus

decreasing nitrogen levels in the effluent.

ª 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic ammonium oxidation (ANAMMOX) is a biochem-

ical process that converts ammonium to N2 by using nitrite as

rch Center for Innovativersity, Guangzhou 510275(F. Meng).

rved.

the electron acceptor (Jetten et al., 2001; Strous et al., 2006).

The ANAMMOX process has been implemented in various

full-scale applications for treating ammonium-rich waste-

water, such as sludge digestion water and industrial

Environmental Technology (SHRCIET), School of Environmental, PR China. Tel.: þ86 20 39335060; fax: þ86 20 84110267.

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 1 83

wastewater (Joss et al., 2009; van der Star et al., 2007; Wett,

2007; Zhang et al., 2011). It can also perform well at low tem-

peratures due to its very high sludge content (Hu et al., 2013;

Wang et al., 2013). Compared with conventional nitrogen

removal processes, the ANAMMOX process can achieve

considerably high nitrogen removal rates (NRR) (Tang et al.,

2011; Tsushima et al., 2007b) with no demands for oxygen

and organics (van Dongen et al., 2001). However, the ANAM-

MOX process requires a very long start-up time if proper

seeding sludge is not available because of the very low growth

rate of ANAMMOX bacteria (Strous et al., 1998). Thus, the loss

of free bacteria should be avoided. An easy and reliable start-

up approach for the ANAMMOX process is needed for its

practical application. In this case, membrane bioreactors

(MBRs), which can effectively retain low-growth biomass

(Trigo et al., 2006, van der Star et al., 2008; Wang et al., 2009),

could provide a solution. However, frequent membrane

fouling and the high investment costs of MBRs hinder their

application to ANAMMOX startup (Trigo et al., 2006, Wang

et al., 2009).

In addition to MBRs, granulation and attached-growth re-

actors also aid in the enrichment of ANAMMOX bacteria to

high concentrations (Tang et al., 2011)(Ni et al., 2012;

Vlaeminck et al., 2008, 2009; Winkler et al., 2011, Winkler

et al. 2012). The stratified distribution of both substrate and

bacteria within the biofilm or granules facilitates cell prolif-

eration and pollutant removal (Ni et al., 2009; Shi et al., 2013;

Volcke et al., 2012). A number of studies have demonstrated

that granule- or biofilm-based reactors achieved high NRR

(Fujii et al., 2002; Tang et al., 2011; Tsushima et al., 2007b), e.g.,

74.3e76.7 g-N/(L.d) in a UASB reactor (Tang et al., 2011) and

26 g-N/(L.d) in a biofilm reactor (Tsushima et al., 2007b).

However, the effluent of current ANAMMOX reactors still

contains nitrogen up to hundreds of mg-N/L, as reported in

previous studies (Tsushima et al., 2007b; van der Star et al.,

2007). In addition, in the biofilm- or granule-based reactors

bacteria wash-out can occur, when biofilm/granule fragments

are formed during an unstable operation period (Trigo et al.,

2006). Therefore, it is necessary to decrease the levels of

nitrogenous species in the effluent or to prevent biomass loss

Fig. 1 e Schematic illustration of the reactor (A) and the working

ANAMMOX bacteria attached on or within the nonwoven fabrics

to pass through the ANAMMOX biofilm and the nonwoven fabric

to that used in conventional MBRs.

during the long-term operation by optimizing current

ANAMMOX reactors and/or developing new processes.

Because biomass aggregates display higher density and

thickness than flocculent sludge and these reactors have

lowermixing intensity, the influx of pollutants and out-flux of

metabolic products through the biomass aggregates could be a

rate-determining step for pollutant removal (Gonzalez-Gil

et al., 2001), particularly for the ANAMMOX process with

high reaction rates. A previous study has reported that a

thicker concentration boundary layer (50e800 mm) reduces the

mass transfer rate up to 50% in the biofilm (Manz et al., 2003).

Considerable research has been devoted to improving reactor

performance, including attempts to increase shear stress

(Beyenal and Lewandowski, 2002; Brito and Melo, 1999; Manz

et al., 2003) and control biofilm thickness (Hwang et al.,

2010). The positive effect of water recirculation on the

ANAMMOX process in UASB reactors could be partially

attributable to the increased shear stress it creates (Jin et al.,

2012). Nevertheless, the previously reported strategies were

mainly focused on the improvement of diffusion-driven

removal (Beyenal and Lewandowski, 2002; Brito and Melo,

1999; Hwang et al., 2010; Manz et al., 2003). Importantly, a

great portion of nitrogenous species may escape from the

reactor during the discharge of treated wastewater. We can

expect that preventing the escape of nitrogen species and

maximizing their diffusion within biofilm matrix hold great

potential for improving the performance of the granule- or

biofilm-based ANAMMOX reactors.

This study proposes to integrate the advantages of con-

ventional MBRs and biofilm reactors by developing a novel

single biofilm reactor that uses a filter of nonwoven fabric to

not only serve as substratum but also act as a membrane-like

liquid/solid separator. Compared with the ANAMMOX biofilm

reactors described in previous studies (Fujii et al., 2002; Gong

et al., 2007; Tsushima et al., 2007b), this reactor is novel

because all the nitrogenous species in the reactor bulk must

pass through the biofilm matrix before being discharged.

Thus, the biofilm could act as a permeable reactive biobarrier,

which contributes to nitrogen removal during water

discharge. In addition, similar to conventional MBRs, this

mechanism of the ANAMMOX biofilm in the reactor (B). The

. During operation, the water and nitrogenous species have

. The enforced water movement in this reactor was similar

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 184

reactor can avoid biomass loss, hopefully shortening the

startup period. In this study, a lab-scale reactorwas developed

and operated for one year to evaluate the performance of this

novel ANAMMOX biofilm process. The concentration differ-

ences of nitrogenous species between the reactor bulk liquid

and reactor filtrate were examined to evaluate the enhanced

removal of nitrogen attributable to the permeable reactive

biobarrier. The effect of the operating flux on the nitrogen

removal and the bacterial community of the biofilm were also

investigated in this study.

Fig. 2 e Performance of the reactor in (A) ammonium

removal, (B) nitrite removal and (C) NRR. The NRR of the

reactor bulk (the curve marked by solid green circles) was

calculated based on the nitrogen levels in the feed

wastewater and the reactor bulk. The NRR of the reactor

filtrate (the curve marked by hollow circles) was calculated

based on the total removed nitrogen in the whole reactor,

i.e., it was the total NRR of the reactor. The enhancement

factor in Fig. 2C represents the contribution of the reactive

barrier process (the differences between the NRR of reactor

bulk and filtrate NRR) to the total NRR. (For interpretation of

the references to colour in this figure legend, the reader is

referred to the web version of this article.)

2. Experimental procedures

2.1. Reactor setup

Similar to our previous study (Meng et al., 2013), nonwoven

fabric sheets (25 � 19 � 0.6 cm) with a mesh size of approxi-

mately 0.1mmwere used as the biofilm carriers in the reactor,

as shown in Fig. 1. Before being installed in the reactor, the

nonwoven fabrics were fixed onto filtration modules. The

modules functioned as both membrane-like separators and

biofilm carriers. A pump enabled the reactor bulk in the

reactor to pass through the modules and allowed the biofilm

aggregates attached to the modules (see Fig. 1b). In this study,

five sets of modules were installed in a reactor with an

effective volume of 15 L (see Fig. 1a). The modules provided a

total surface area of 0.23 m2 and occupied an intrinsic volume

of 3.12 L. The space between themoduleswas 4 cm. During the

entire operation, the reactor was covered with black cloth to

prevent exposure of the ANAMMOX bacteria to light to avoid

the proliferation of phototrophic microbes. Neither relaxation

nor backwashing was performed for the nonwoven modules

during the entire operation period. The liquid level was

maintained by a liquid-level controller. The transmembrane

pressure (TMP) of the nonwoven modules, including the

nonwoven fabrics and filtration unit, was monitored using a

vacuum gauge. It was found that the reactor biomass was

mainly present in the form of attached growth on or within

the nonwoven fabrics.

2.2. Reactor operation

The reactor was initially inoculated with seeding sludge (ca.

1.6 g in dry weight) from an ANAMMOX-based reactor oper-

ated at the Dalian University of Technology. The influent was

prepared by combining (NH4)2SO4 and NaNO2 at a molar ratio

of 0.5. The concentrations of ammonium and nitrite nitrogen

in the influent were increased gradually during operation

from 25 to 500 mg-N/L by changing the levels of ammonium

and nitrite in the feed wastewater (see Fig. 2 and Table S1).

Trace elements, with modifications after Van de Graaf et al.

(1996), were added in the influent to aid in the growth of

ANAMMOX bacteria. The influent compositions are shown in

Table S1 in the Supplementary Materials (SM) file. The flux

through the nonwovenmoduleswas increased gradually from

2.15 to 4.3 L/m2h (LMH), resulting in a decrease in the HRT

from 24 to 12 h. Due to the changes in the influent nitrogen

concentration and reactor HRT, the corresponding nitrogen

loading rate (NLR) was increased from 0.05 to 2.0 kg-N/(m3 d).

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 1 85

Considering the consumption of inorganic carbon for the

proliferation of ANAMMOXbacteria, NaHCO3was added to the

influent water, which also maintained the pH of the influent

in the range of 7.5e7.8. The reactor temperature was main-

tained at 34 � 1 �C using a heater. To speed up the start-up of

the reactor, the reactor was purged with N2 gas in the opera-

tion period of day 0e60, which can maintain a dissolved ox-

ygen (DO) concentration of less than 0.4 mg/L. As the N2 gas

was provided at low rates (<20 L/h) in the form of micro-

bubbles, it did not cause significant biofilm detachment.

From day 61 on, the reactor remained at a low DO level

(0.2e0.4 mg/L) automatically without any N2 sparging, likely

due to the significant oxygen uptake by aerobic ammonium-

oxidizing bacteria (AAOB) (see Section 3.4). Details of the

operating parameters are provided in Table S2 in the SM file.

Samples of the influent, reactor bulk and reactor filtrate were

collected twice per week for the analysis of ammonium and

nitrite. And, the filtrate samples were taken from the effluent

outlet rather than the collection tank, which can avoid nitro-

gen loss during the storage. Thus, the differences of nitrogen

levels in reactor bulk and reactor filtrate can reveal the role of

the reactive barrier on nitrogen removal.

2.3. Short-term tests using the reactor

After 380 days of long-term operation, the effect of the flux on

the removal of nitrogen by the biofilm was investigated

through short-term (oneweek) tests. Fluxes of 4.3, 8.6 and 17.2

LMH were applied, with corresponding HRTs of 12, 6 and 3 h,

respectively. To maintain a constant NLR of 2.0 kg-N/(m3 d),

the concentrations of nitrite/ammonium in the influent were

maintained at 500, 250 and 125 mg-N/L under the respective

fluxes of 4.3, 8.6 and 17.2 LMH. The temperature, pH and trace

element concentrations in all these tests were kept the same

as those used during the long-term operation. During each

test run, samples were collected bi-hourly between 9:00 and

21:00 to monitor the concentrations of ammonium and nitrite

in the influent, reactor bulk and reactor filtrate. After

completion of these runs, the reactor was returned to the

normal operating conditions, and it returned to normal

operation in 1e3 weeks.

2.4. Filtration test

To compare the filterability of ANAMMOX sludge with con-

ventional sludge, two sets of filtration test were conducted in

this study. Sludge samples were taken from the ANAMMOX

reactor and a local municipal wastewater treatment plant

(WWTP). Prior to filtration tests, the sludge samples were

washed with pure water to remove dissolved organic matter.

Subsequently, the sludge concentration was diluted to about

3800 mg/L using pure water. Then, the filterability of these

sludge samples was conducted in a filtration cell (MSC300,

Mosu Co, Shanghai, China) in a steady pressure mode. Pre-

cleaned membranes (0.22-mm, PVDF, Xinya, Shanghai, China)

were used for the filtration tests. The membranes have a

diameter of 80 mm and a filtration area of 0.005 m2. The

filtration pressure was provided with a water head drop of

70 cm. To avoid automatic settling of the sludge particles, the

filtration cell was continuously stirred at a speed of 100 rpm.

During the filtration process, pure water was continuously

added to the filtration cell tomaintain a steady liquid level and

thus a steady water head drop. The accumulative filtrate

volume was recorded regularly.

2.5. DNA Extraction, PCR amplification and phylogeneticanalysis

Genomic DNA was extracted from the centrifuged (5 min

at 13,000 g, ca. 0.3 g of wet biomass) samples using the

ultraClean Soil DNA kit (MoBio, Solana Beach, Calif.) ac-

cording to the manufacturer’s protocol. The complete 16S

rRNA genes were amplified in triplicate with two primers of

27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1492R (5-

GGTTACCTTGTTACGACTT-3) (Dojka et al., 1998). The hy-

drazine synthase b subunit (hzsB) genes in the ANAMMOX

bacteria were amplified with hzsB-396F (ARGGHTGGGGHA-

GYTGGAAG) and hzsB-742R (GTYCCHACRTCATGVGTCTG),

both of which have been demonstrated to be very specific and

sensitive primers for targeting ANAMMOX bacteria (Harhangi

et al., 2012; Park et al., 2010; Schmid et al., 2005; Wang et al.,

2012). In addition, the amoA genes in the AAOB were ampli-

fied with amoA-1F (GGGGTTTCTACTGGTGGT) and amoA-2R

(CCCCTCKGSAAAGCCTTCTTC) (Rotthauwe et al., 1997).

The PCR programs are shown in the SM file of this manu-

script (Table S3). The quality of the PCR products was evalu-

ated with agarose gel electrophoresis (see Figure S1), and then

the products were purified with the QIAquick PCR purification

kit (Qiagen Crawley, UK). Afterwards, they were cloned into

the pMD18-T vector (Takara, Dalian, China). Randomly

selected clones (120e160 for each library) containing correctly

sized inserts were sequenced at a commercial facility (BGI,

Shenzhen, China). Chimeric sequences were identified and

excluded from subsequent analysis as described previously

(Huang et al., 2004). Sequences in each clone library were

binned into operational taxonomic units (OTU) at a similarity

of>97%. Phylogenetic analyseswere performed using the ARB

software package (Ludwig et al., 2004).

2.6. Chemical analyses

Prior to chemical analysis, all samples were filtered through

0.45-mm membrane filters (PVDF, Xinya, Shanghai, China) to

remove particulates, including free bacteria. The concentra-

tions of ammonium, nitrite, total nitrogen and total sus-

pended solids (TSS) were determined according to the

Standard Methods (APHA, 1995). The pH and DO were

measured using a pH meter (PHS-3D, Shanghai Rex Instru-

ment Co., Shanghai, China) and a DO meter (CellOX 3310i,

WTW Co., Munich, Germany), respectively. The turbidity of

the reactor filtrate was determined using a HACH Turbidim-

eter (2100N, HACH Co., Loveland USA).

3. Results and discussion

3.1. Reactor performance

The reactor was operated over a period of 380 days. The

reactor achieved satisfactory nitrogen removal (on average,

0.00

0.20

0.40

0.60

0.80

1.00

4.3 LMH (500 mg-N/L)

8.6 LMH (250 mg-N/L)

17.2 LMH (125 mg-N/L)

NR

Rkg

-N/m

3 ·d

AmmoniumNitrite

(A)

0%

5%

10%

15%

20%

25%

30%

35%

4.3 LMH (500 mg-N/L)

8.6 LMH (250 mg-N/L)

17.2 LMH (125 mg-N/L)

Con

tribu

tion

to th

e N

RR

AmmoniumNitrite

(B)

0%

10%

20%

30%

40%

50%

60%

70%

80%

4.3 LMH (500 mg-N/L)

8.6 LMH (250 mg-N/L)

17.2 LMH (125 mg-N/L)

Perc

en.o

f the

rem

oved

Nin

reac

tor s

uper

nata

nt

Ammonium

Nitrite

(C)

Fig. 3 e Performance of the reactor at different imposed

flux: (A) total NRR of the reactor at different fluxes, (B)

contribution of the reactive barrier process to the total NRR

and (C) percentage of the nitrogenous species in the reactor

reactor bulk removed by the reactive barrier process. The

calculation equations for (B) and (C) are shown in the SI file.

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 186

82% and 84% for ammonium and nitrite, respectively) early in

the operating period (day 0e11), most likely due to the high

abundance of ANAMMOX bacteria in the seeding sludge. Once

good performance was achieved, the concentrations of

ammonium and nitrite in the influent were increased to

250 mg-N/L on the following day (day 12), and the HRT was

decreased from 24 to 12 h on day 20. Despite the increased

NLR, the reactor maintained good nitrogen removal (see

Fig. 2A and B), and the average removal efficiencies for

ammonium and nitrite on days 12e61 were 83% and 96%,

respectively. However, further increases in the NRL from day

62 resulted in higher concentrations of nitrite and ammonium

in the reactor filtrate, especially on days 171e186, when the

concentrations of both ammonium and nitrite in the reactor

filtrate increased to approximately 500 mg/L, indicating

decreased reactor performance. Nevertheless, as shown in

Fig. 2C, the NRR of the reactor increased and then maintained

a good removal rate throughout the remaining operating days,

eventually achieving a rate of 1.6 kg-N/(m3 d). The increased

NRR after day 186 was likely due to the growth of more

ANAMMOX bacteria in the reactor system. Because of the high

attachment propensity of the ANAMMOX bacteria, as well as

the suction applied by the pump, this reactor was dominated

by attached-growth biofilm (41 g at the end of operation) over

the suspended growth sludge (2.2 g at the end of operation).

Interestingly, we observed that the reactor filtrate con-

tained lower levels of both ammonium and nitrite than those

found in the reactor bulk (see Fig. 2A and B). On average, 26.7%

and 48.4% of the remaining ammonium and nitrite in the

reactor bulk were removed when water passed through the

permeable reactive barrier of biofilm. This result implies that

the permeable reactive barrier can significantly improve the

quality of the reactor filtrate, as shown in Fig. 2C. Accordingly,

the enforced water flow in the biofilm matrix contributed to

the NRR significantly, accounting for 11.2% of the total NRR.

This corroborates our previous report that the reactive barrier

process of a biofilm improved nitrate removal in a simulta-

neous nitrification-denitrification (SND) reactor (Meng et al.,

2013). In our reactor, the water flow can force all the pollut-

ants in the reactor bulk to transport into the biofilm before

being discharged. In contrast to the random diffusion that

takes place in conventional biofilm matrix (Beyenal and

Lewandowski, 2002; Brito and Melo, 1999; Hwang et al., 2010;

Manz et al., 2003), the enforced water movement in our

reactor could regulate the diffusion of pollutants within the

biofilmmatrix in a more defined direction, e.g., from the bulk/

biofilm interface to the biofilm/reactor filtrate interface.

Obviously, our results suggest that the biofilm matrix in this

reactor indeed acted as a reactive barrier, which can further

remove some nitrogenous species that was carried by the

water flow. In addition, the advective driven transport of

nitrogenous species within the biofilm could facilitate the

supply of sufficient substrate to the bacteria at the bottom of

the biofilm, thus elevating microbial activity.

The nitrite/ammonium conversion ratio in this reactor was

determined to be 1e1.3 (Figure S3), which is lower than the

theoretical value (i.e., 1.32) but in agreement with values re-

ported previously (Schmid et al., 2000; van der Star et al., 2008).

The relatively low nitrite/ammonium conversion ratio in the

present reactor is most likely due to the presence of AAOB in

the biofilm, as discussed in the following section. The average

pH values of the influent, reactor bulk and reactor filtrate were

determined to be 7.51, 8.07 and 8.17, respectively. As we only

controlled the pH in the influent water, the changes in pH

Fig. 4 e Variations of filtration rates of ANAMMOX sludge

collected form this current reactor and activated sludge

collected from a local municipal WWTP over accumulative

filtration volume. The filtration tests were conducted in a

filtration cell using microfiltration membranes (0.22 mm for

pore size, 0.00524 m2 for filtration area) with a steady

water head drop of 70 cm.

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 1 87

during the reactor operation were attributable to the occur-

rence of the ANAMMOX process. The difference between the

pH of the reactor bulk and that of the reactor filtrate further

revealed the contribution of reactive barrier process to the

ANAMMOX reaction in the biofilm matrix.

3.2. Influence of filtrate flux on reactor performance

To understand the effect of the reactor filtrate flux through the

nonwovenmodules on the reactor’s performance, particularly

on the reactive barrier process, three sets of short-term tests

were conducted as described in the methodology section. The

NLR for these tests was maintained at the same level (2.0 kg-

N/(m3 d) used during normal reactor operation. Fig. 3A depicts

the NRR with respect to ammonium and nitrite in the reactor

during these tests. Clearly, the changes in the flux had little

impact on the NRR, demonstrating that the influence of the

flux on a given NRR (1.5e1.6 kg-N/(m3 d)) could be ignored.

However, the contribution of the reactive barrier process to

the NRR was significantly elevated by increased flux (see

Fig. 3B), e.g., the enhancedmass transfer by the reactor filtrate

drag contributed 22.9% and 29.1% to the ammonium and ni-

trite removal, respectively, when the flux was 17.2 LMH. In

fact, the lower nitrogen concentrations in the tests for higher

fluxes will cause significant decreases in the concentration

gradients in the bulk/biofilm interface, thus lowering the

chance for diffusion-driven removal. Additionally, operation

at a higher flux will inevitably cause physiological responses

as a result of the increased hydraulic drag, which can also

potentially decrease the diffusion-driven removal of nitrogen.

In contrast, the increased water movement of the higher

fluxes enables more nitrogenous species to be transported

through the biofilm matrix, thus resulting in the removal of

some nitrogenous species during permeation (Fig. 3C). Thus,

the reactive barrier process of the biofilmmatrix would play a

more significant role when the reactor was operated with

higher fluxes or lower nitrogen concentrations in the influent

water.

3.3. MBR-related performance of the reactor

The permeability of the filtration modules is of great impor-

tance for attaining sustainable operation of this MBR-like

ANAMMOX reactor. During the entire operation period

under a constant flux, the TMP of the modules was always

below the detection limit (<0.5 kPa), implying that the reactor

had not become fouled. Even when it eventually fouls, the

reactor can be backwashed by the reactor filtrate, as it is a

macro-filtrationMBR using coarse filters (Bai et al., 2010; Meng

et al., 2013), and its permeability can be recovered quickly

(Meng et al., 2013). Hence, this new type of reactor is poten-

tially feasible for long-term operation of the ANAMMOX pro-

cess. Three major factors most likely account for the

constantly low TMP. First, the biofilm formed by the ANAM-

MOXbacteria (i.e., autotrophic bacteria) is expected to bemore

permeable than conventional biofilms that are dominated by

heterotrophic bacteria, likely because the ANAMMOX bacteria

tend to form granules inside the biofilm matrix (see Figure S2

and S3). Ni et al. (2010) also found ANAMMOX granules having

a high compactness in a nonwoven biofilm reactor; they

attributed this phenomenon to the high abundances of

extracellular polymer substrates (EPS) of ANAMMOX bacteria.

Fig. 4 shows the profile of filtration rates of ANAMMOX sludge

and activated sludge from a WWTP plant over filtration vol-

ume. It can be seen that the ANAMMOX sludge had lower

filtration rates than the WWTP sludge in the initial filtration

volume (0e150 mL). However, in the following filtration pro-

cess (150e600mL) the filtration rates of the ANAMMOX sludge

seemed to keep at a steady level; in contrast, those of the

WWTP sludge decreased continuously, with lower filtration

rates than those of ANAMMOX sludge. It suggests that the

ANAMMOX sludge had a strong propensity to maintain high

and steady permeability during the long-term filtration. Sec-

ond, because of the high NRR of ANAMMOX process, the

occurrence of ANAMMOX reactions could yield much more

considerable amounts of N2 gas bubbles than the conven-

tional denitrifying processes treating municipal wastewater;

the in-situ release of gas bubbles from the biofilmmatrix to the

reactor bulk could make the biofilm porous and permeable.

Third, the non-wovenmoduleswere operated at amuch lower

flux (4.3 LMH) than conventional MBRs (10-20 LMH). In our

previous study (Meng et al., 2013), the nonwoven modules

were operated at the same flux (4.3 LMH) in the same reactor,

but they were backwashed with tap water once every 20e30

days when treating synthetic municipal wastewater. Thus,

the lower fouling rates of this ANAMMOX reactor could be

attributable to the first two reasons, i.e., high permeability of

the ANAMMOX biofilm and in-situ release of N2 gas bubbles

during the ANAMMOX reaction.

The turbidity of the reactor filtrate decreased from 16.4 to

approximately 5.9 NTU during the initial 12 h and then sta-

bilized at an average of 3.0 NTU. During the whole operation,

the suspended biomass in the reactor bulk was in the range of

120e180 mg/L, while that in reactor filtrate was always below

6.5 mg/L. The low levels of turbidity and biomass amounts in

the reactor filtrate suggest that the reactor could effectively

avoid biomass wash-out. A major difference between our

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 188

reactor and previous MBR reactors is that most of the free

bacteria in the mixed liquor were attached to the nonwoven

fabric as a result of the hydraulic drag by the suction pump.

The low concentration of suspended bacteria (120e180 mg/L)

could have contributed to the low turbidity and free bacteria

observed in the reactor filtrate. In addition to the improved

nitrogen removal, therefore, the prevention of biomass wash-

out is another important feature of this reactor which facili-

tates the start-up of the ANAMMOX process.

3.4. Bacterial community compositions

The 16S rRNA gene clone library of all the bacteria in the

biofilm was constructed using a universal primer set (27F and

1942R). In total, 68 clones were effectively sequenced, and

they were grouped into 23 OTUs according to the requirement

of >97% similarity (see Figure S4). These OTUs were affiliated

with six phyla: Armatimonadetes, Proteobacteria, Chloroflexi,

Planctomycetes, Chlorobi and Bacteroidets. Similar to previous

findings (Li et al., 2009; Strous et al., 2006; Tsushima et al.,

2007a), the current results reveal the symbiosis of ANAM-

MOX bacteria (i.e., Planctomycetes) with other phyla in the

reactor. In fact, the Chloroflexi-like filamentous bacteria are

assumed to play an important role in the formation of biofilm

or granules (Cho et al., 2010; Li et al., 2009). Among the three

OTUs in the phylum Planctomycetes, however, only one OTU

(Candidatus Kuenenia-like species) with three clones was

identified to be the recognized ANAMMOX bacteria species.

Additionally, the phylum Planctomycetes had a lower abun-

dance than expected (14.3%). These results imply that the 16S

rRNA used to sequence universal bacteria is not sensitive or

effective for the detection of ANAMMOX culture (Li et al., 2009;

Schmid et al., 2000, 2008).

In an attempt to improve the sensitivity and specificity of

the sequencing, a clone library based on the amplification of

hzsB functional genes, which are very specific to ANAMMOX

bacteria (Harhangi et al., 2012; Strous et al., 2006; Wang et al.,

Fig. 5 e Phylogenetic tree of ANAMMOX bacteria based on ampli

are shown in the brackets.

2012), was constructed. As shown in Fig. 5, 115 clones grouped

into 7 OTUs were detected by the amplification of hzsB genes.

All the OTUs had high sequence similarity (94%e100%) to the

hzsB genes of the known ANAMMOX species (see Table S4). By

using the ANAMMOX hzsB gene primer, in addition to primers

from the Candidatus Kuenenia-like species (83/115, 72.2%) that

were found in the clone library, two additional species, Can-

didatus Jettenia-like species (28/115, 24.3%) and Candidatus

Brocadia-like species (4/115, 3.5%), were detected. These re-

sults also demonstrate the dominance of Candidatus

Kuenenia-like species in the reactor. Compared with Candi-

datus Brocadia-like species, the Candidatus Kuenenia-like

species have a higher affinity with nitrite (Strous et al.,

1999). Recently, van der Star et al. (2008) reported the shift of

functional ANAMMOX bacteria from Candidatus Brocadia-like

species to Candidatus Kuenenia-like species during the culti-

vation of an MBR with an SRT of 16 days. The Candidatus

Kuenenia-like species is able to remove nitrate from the

wastewater via the dissimilatory nitrate reduction process

(Kartal et al., 2007). Based on the amplification of amoA func-

tional genes in this study, we also observed the presence of

AAOB falling under the phylum N. europaea (see Figure S5),

which can often be found in wastewater treatment plants

with high NLR (Ahn et al., 2008; Kotay et al., 2013). The AAOB

activity was sufficiently high to remove most of the oxygen

transferred into the bulk liquid via air/liquid exchange, so that

the DO concentrationswere at levels between 0.2 and 0.4mg/L

during the long-term operation. The occurrence of nitritation

by AAOB could also explain why a lower nitrite/ammonium

conversion ratio than the theoretical value was observed

(1.32).

3.5. Implications and research needs of this study

This study developed a new reactor based on both biofilm

reactors and MBRs. To our knowledge, this is the first study to

incorporate MBR-like operation into an ANAMMOX biofilm

fication of the hzsB gene. The clone numbers of a given OTU

wat e r r e s e a r c h 5 8 ( 2 0 1 4 ) 8 2e9 1 89

reactor in an attempt to improve nitrogen removal by using

the biofilm as a reactive barrier. In this reactor the advective

transport of nitrogenous species in the biofilm matrix can be

expectedly enhanced. In the reactor the nonwoven fabrics,

which are much cheaper than the commercially available

micro-filtration membranes used in conventional MBRs, play

a dual role, integrating membrane-like separators and

biomass carriers in a single reactor. The biofilms that form

also serve a dual function, acting as bio-degraders and sec-

ondary dynamic membranes. These combinations can not

only simplify reactor design but also enhance performance.

Crucially, in addition to their good rejection performance for

free bacteria, the nonwoven modules can maintain a high

permeability during the entire operation, possibly because of

in-situ release of N2 gas bubbles and the high permeability of

the biofilm matrix. Note that the maintenance of an unfouled

state is an important prerequisite for sustainable operation of

such a reactor. Unlike conventional MBRs, chemical cleaning

is not recommended for this type of reactor, as it can kill the

microbes in the biofilm.

In brief, the reactor showed strong economic benefits (use

of low-cost nonwoven fabrics) and technical merits (improved

nitrogen removal, membrane-like separation and non-fouling

operation) for practical applications in the future. However,

this study was based on a single ANAMMOX process treating

synthetic wastewater that was prepared with nitrite nitrogen

and ammonium nitrogen. In the future, this reactor should be

extended to nitritation-ANAMMOX processes (Joss et al.,

2011), such as oxygen-limited autotrophic nitrification/deni-

trification (OLAND) (De Clippeleir et al., 2012; Vlaeminck et al.,

2009). Additionally, further investigation focussing on

microelectrode-assisted measurements and the modelling of

nitrogen transport/transformation within the biofilm matrix

should be conducted to better understand the role of perme-

able reactive biobarrier in nitrogen removal.

4. Conclusions

This study reported on an ANAMMOX biofilm reactor that

uses the biofilm as permeable reactive barrier for improving

the removal of nitrogen species. The main conclusions of this

work can be drawn as follows:

(1) The reactor achieved NNR of about 1.6 kg-N/(m3 d). The

reactive barrier process removed 27% of the ammonium

and 48% of the nitrite in the reactor bulk during their

transport through the biofilm, which finally contributed

ca. 11% to the total NRR during the long-term reactor

operation.

(2) The contributions of the reactive barrier process for

nitrogen removal depended strongly on either nitrogen

concentrations in reactor bulk or the imposed fluxes.

The reactive barrier process played a more important

role at lower nitrogen concentrations or higher fluxes.

(3) The nonwoven modules as well as the attached biofilm

avoided the loss of free bacteria effectively. Because of

the higher permeability of the ANAMMOX biofilm and

in-situ release of N2 gas bubbles, the filtration modules

maintained a non-fouling state during the entire oper-

ation period.

(4) The ANAMMOXculture in the biofilmwas dominated by

Candidatus Kuenenia-like species (72.2%), followed by

Candidatus Jettenia-like (24.3%) and Candidatus Brocadia-

like species (3.5%).

Acknowledgements

This study was supported by the National Natural Science

Foundation of China (No. 21107144), the Natural Science

Foundation of Guangdong Province (No. S2011010001507) and

a project of Science and Technology ZhuJiang New Star in

Guangzhou city (2012J2200045). Many thanks to Prof. Fenglin

Yang at Dalian University of Technology for supplying the

seeding biomass.

Appendix A. Supplementary data

Supplementary data related to this article can be found at

http://dx.doi.org/10.1016/j.watres.2014.03.049.

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