Bioresource Technology 104 (2012) 73–80

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Bioresource Technology

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Impact of reactor configuration on anammox process start-up: MBR versus SBR

Yu Tao a, Da-Wen Gao a,b,⇑, Yuan Fu a, Wei-Min Wu c, Nan-Qi Ren a

a State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, Chinab School of Forestry, Northeast Forestry University, Harbin 150040, Chinac Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 July 2011Received in revised form 13 October 2011Accepted 16 October 2011Available online 20 October 2011

Keywords:Anaerobic ammonium oxidationSequencing batch reactorMembrane bioreactorAnammox activityBiological nitrogen removal

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.10.052

⇑ Corresponding author at: State Key Laboratory oEnvironment, Harbin Institute of Technology,Tel./fax: 86 451 86289185.

E-mail addresses: gaodw@hit.edu.cn, dawengao@g

Anaerobic ammonium oxidation (anammox) is an energy saving biological nitrogen removal processwhich was limited to slow growth rate of anammox bacteria during start-up period. This study investi-gated the start-up of anammox process by a laboratory sequential batch reactor (SBR) for 218 days andsubsequently modified the reactor as a membrane bioreactor (MBR) for 178 days. Modification of a SBR asMBR with installation of an external membrane module resulted in acceleration of specific anammoxactivity by 19 times. The acceleration of specific anammox activity with MBR was further confirmedby starting-up another MBR for a 242 day period. Molecular microbial analyses showed that Candidatus‘‘Brocadia anammoxidans’’ and Candidatus ‘‘Kuenenia stuttgartiensis’’ were the dominant species in theinocula and biomass developed in the reactor. The start-up with MBR appeared to be more effective thanSBR for the enrichment of anammox bacteria due to high sludge retention property of MBR configuration.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Excessive production of anthropogenic nitrogen causes severeeutrophication of terrestrial and aquatic systems and has alreadybeen of global concern (Galloway et al., 2008). Many countrieshave strictly regulated the discharge of nitrogen-rich wastewaterinto aquatic system. For wastewater treatment plants (WWTPs),more stringent discharge standard calls for an update of nitrogenremoval technologies to replace the conventional one (e.g. full orshortcut nitrification/denitrification), which costs high investmentand energy. Anammox process provides an attractive option for to-tal nitrogen (TN) control (Kartal et al., 2010).

Anammox process was observed in a pilot denitrifying fluidized-bed reactor (Mulder et al., 1995) and has since been successfully ap-plied to treat various types of ammonia-rich wastewater (Strouset al., 1997; Liang et al., 2009; Joss et al., 2009). Bacteria responsiblefor anammox are related to Planctomycetes (Strous et al., 1999).These organisms can couple the oxidation of ammonium with thereduction of nitrite to nitrogen gas (N2) as the terminal product(Van de Graaf et al., 1995). Anammox bacteria grow at a slow rate(a common opinion is doubling time 8–11 days) (Strous et al.,1998; van der Star et al., 2008), and are extremely difficult to growin vitro.

Some factors have been found to be able to influence the start-upof anammox process including hydraulic retention time (HRT), dis-

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f Urban Water Resource andHarbin 150090, China.

mail.com (D.-W. Gao).

solved oxygen (DO), inoculum, temperature, wastewater composi-tion and nitrogen compound concentration (Tang et al., 2010a;Tsushima et al., 2007). Recently, reactor configuration was alsodemonstrated to have a significant impact on cultivation of anam-mox bacteria (Hu et al., 2010).

A reactor configuration with high biomass retention is essentialfor anammox process due to their slow growth rate. Membranebioreactor (MBR) and sequencing batch reactor (SBR) are widely-ap-plied bioreactors for wastewater treatment (Shannon et al., 2008;Gao et al., 2010). MBR can prevent product inhibition and the out-flow of suspended cells (Jagersma et al., 2009). Several studies haveproved that a submerged MBR was an excellent tool for enrichingslow-growing microorganisms such as methanotrophic archaea(Meulepas et al., 2009) and anammox bacteria (van der Star et al.,2008). However, the anaerobic condition of a submerged MBR couldbe interrupted if membrane modules are frequently replaced due tobiofouling (Gao et al., 2009), which could harm anammox bacteria(Meulepas et al., 2009). An external MBR (or EMBR) could avoid suchinterruption. To date, studies that investigated the start-up of anam-mox process by using an external MBR are very limited and the com-parison of SBR versus MBR in anammox start-up is also rare.

The objective of this study is to assess the start-up of anammoxprocess with EMBR versus SBR. The performance of these reactorswere compared were based on (a) nitrogen (ammonium, nitriteand nitrate) removal efficiency; (b) specific anammox activity(SAA) of the biomass enrichment in the two reactors; (c) fluores-cence in situ hybridization (FISH) analysis for anammox bacteria;(d) microbial community analysis; and (e) activity variations of sidepopulations.

74 Y. Tao et al. / Bioresource Technology 104 (2012) 73–80

2. Methods

2.1. Reactor design

Two same sized reactors were used in this study. Reactor R1was started as a SBR system and operated for 218 days and subse-quently was modified as an EMBR for 178 days named as R2. Whenthe reactor was operated as an SBR, no membrane module and re-cycle pump was installed (Fig. 1). Reactor R3 was started as anEMBR and operated for 242 days. The reactors were made ofplexiglass with the identical cylindrical shape and effective volume(3.0 L). Fig. 1 shows the system design of EMBR. A mechanical stir-rer kept completely mixing the sludge. The effective volume ofmembrane module tube was 188 mL and the flux of recyclingpump was 170 mL/min. Sludge mixture stayed only 1.1 min (onaverage) in the module. Polyethylene (PE) hollow fiber sheets(Mitsubishi Rayon Co. Ltd., Tokyo, Japan) were used, having a meanpore size of 0.4 lm. The membrane surface is 0.03 m2 and the max-imum flux is 10 L day�1. In order to continually measure the trans-membrane pressure (TMP) and actual flux of the EMBR, electronicpressure and flux sensors (JYB-KO-HAG and JYB-KB-LAG, BeijingCollihigh Sensor Technology Center, China) were equipped. A PCcomputer was used to collect and analyze data. To prevent lightpenetration and the growth of phototrophic microorganisms, thereactors were entirely covered with a black shade.

2.2. Inoculation

Reactor R1 was inoculated with 500 mL of stored aerobic gran-ular sludge from a 12 L laboratory-scale SBR treating syntheticwastewater with glucose substrate (Liu et al., 2010). The volatilesuspended solid (VSS) of inoculated aerobic granules was0.3 g L�1. After 218-day operation, R1 was modified as EMBR andequipped with membrane module, recycling pump, and pressureand flux sensors as an EMBR (R2). Reactor R3 was inoculated with3 L of activated sludge (an initial VSS content of 5 g L�1) from aWWTP.

2.3. Synthetic medium

R1, R2 and R3 were fed with synthetic medium. The source ofammonium and nitrite was prepared using NH4Cl and NaNO2. Thenitrogen loading of the two reactors were gradually increased bysupplementing NH4Cl and NaNO2 to influent. The composition ofother mineral medium was 10 mM inorganic carbon (KHCO3),

Fig. 1. Schematic diagram of the anammox system with external membrane (EMBR). Theand other relevant components. The EMBR module tube had a diameter of 40 mm and

0.074 mM phosphor (KH2PO4), 0.24 mM magnesium (MgSO4�7H2O), 0.034 mM calcium (CaCl2�2H2O), 0.040 mM iron (FeS-O4 + EDTA) and 1.25 mL L�1 of trace elements solution. The traceelement solution contained (g L�1) (adapted from Van de Graafet al., 1996): H3BO4, 0.014; MnCl2�4H2O, 0.99; CuSO4�5H2O, 0.25;ZnSO4�7H2O, 0.43; NiCl2�6H2O, 0.19; NaSeO4�10H2O, 0.21; Na-MoO4�2H2O, 0.22; and CoCl2�6H2O, 0.24. Inert gas (95% Ar + 5%CO2) was purged into the influent tank during the preparation ofmedium to remove the oxygen (influent DO < 0.08 mg L�1). ThepH of influent was 7.7–8.3.

2.4. Reactor operation

The operational conditions of three reactors are summarized inTable 1. The HRT of R1, R2 and R3 were 48 h. The reactors wereequipped with a temperature controller to maintain the tempera-ture of 31–32 �C. There was no excess sludge removed from thereactors during the whole experimental trial, except for samplingfor MLSS (25 mL), FISH (1.5 mL) and SAA analysis (10 mL). An R1cycle consisted of 1 h of continuous supply of medium, 46.25 h ofmixing, a settling period of 0.5 h, and 15 min to draw off liquidfrom above the settled sediment and biomass. Inert gas was purgedto R1 several minutes before and after the filling period to driveaway most oxygen left in R1. R2 and R3 were operated for 178and 242 days, respectively, and the membrane sheets were almostbimonthly changed due to membrane fouling (Gao et al., 2009).Four membrane modules that were exactly the same were usedalternately. Old membranes were replaced when the TMPexceeded 50 kPa.

2.5. Analytical methods

Ammonium, nitrite, nitrate, MLVSS were measured according tostandard methods (APHA, 1998). Total organic carbon (TOC) andTN were determined with a Shimadzu TOC-VCPN-6000 analyzer.Parameters including pH and DO were tested by Handheld Multi-Parameter Instruments (pH/Oxi 340i, WTW, Germany).

2.6. Batch tests for specific activities

Batch tests were conducted for the determination of specificactivities for the removal of ammonium, nitrite and nitrate tocharacterize the activities of different trophic groups of bacteriainvolved in nitrogen cycle in the biomass. The consumption rateof ammonium-nitrite was used for the calculation of the activity

SBR system used was the same reactor but without EMBR module, recycling pumpheight of 150 mm.

Table 1Operational condition of the anammox system.

Operational parameter Value

R3 R1 R2

Temperature 31 ± 1 �CpH of influent 8.0 ± 0.3pH in reactor 7.5 ± 0.5 7.3 ± 0.4 7.1 ± 0.7DO of influent <1.0 mg L�1

DO in reactor <0.08 mg L�1 <0.06 mg L�1 <0.08 mg L�1

TOC of influent 0TOC in reactor 7.8–85.2 mg L�1 5.5–53.7 mg L�1 1.1–5.2 mg L�1

HRT 2.0 daysRetention time of sludge in the membrane module 1.5 min – 1.5 minEffective life-span of the membrane 31–37 days – 26–35 days

Y. Tao et al. / Bioresource Technology 104 (2012) 73–80 75

of anammox bacteria, the ammonium removal rate under aerationfor AOB, the nitrite depletion under aeration for NOB, the nitratedepletion under anaerobic condition with acetate for DNB, andthe conversion of nitrate to ammonium under anaerobic conditionfor DNRA. Microbial biomass from the reactors was washed withphosphate buffer (pH 7.8) which was previously sparged with Argas. The biomass was then transferred into 100 mL serum bottlesand supplemented with respective substrate solution to achieve5 mM concentration of ammonium, nitrite or nitrate, and incu-bated at 35 �C. The concentrations of each nitrogen compoundwere measured after incubation (normally 12 h for anaerobic pro-cess and 4 h for aerobic process). Triplicates were used in thisstudy. The specific rates were calculated based on the substrateconsumption and the amount of biomass added.

2.7. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNAanalyses

The biomass for DGGE analysis were sampled from R1 at day 1,53, 117, 163 and 212, respectively, and from R3 at day 1, 26, 77,133, 196 and 240, respectively. Specific bacterial primer GC-BSF338 (primer 338 plus a GC clamp attached to its 50 end) and areverse universal primer BSR518 (Shanghai Sangon Biology Engi-neering Technology and Services Co. Ltd, China) were used to am-plify bacterial 16S rDNA. The nucleotide sequence of the primerswas as follows: Primer GC-BSF338, 50-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGGA-GGCAGCAG-30; primerBSR518, 50-ATTACCGCGGCTGCTGG-30. The PCR cycle was startedwith a 5 min denaturation step at 94 �C, followed by 35 cycles ofdenaturation for 30 s at 94 �C the annealing temperature of thetouch-down PCR was 65–56 �C, and concluded by a 30 s extensionat 72 �C and a final extension at 72 �C for 8 min. PCR products wereverified in 1% agarose gel. DGGE analysis of PCR products was per-formed with a BioRad D-Code System (BioRad Laboratories, Missis-sauga, Ontario, Canada). PCR samples were concentrated and300 ng were loaded onto an 8% (w/v) polyacrylamide gel contain-ing a 30–60% gradient of denaturant (100% denaturant correspondto 5.6 M urea and 32% (v/v) deionized formamide). Bands of inter-est were reamplified, purified and sequenced using the Gel Recov-ery Purification Kit (Watson Biotechnologies Inc., Shanghai, China)according to the manufacturer’s instruction. The DNA sequenceswere determined using the chain termination method in an ABI3730 stretch sequencing system by a commercial service (Sangon,China), and submitted for comparison to GenBank database usingBLAST algorithms.

2.8. Fluorescence in situ hybridization (FISH)

Sludge samples collected from R1, R2 and R3 were fixed in 4%paraformaldehyde (w/v) in phosphate-buffered saline (PBS) at4 �C. After 2 h, the cells were washed three times with PBS and finally

resuspended in a 1:1 (v/v) mixture of PBS and ethanol, and stored at�25 �C. Fixed samples were resuspended in a solution of 200 mg L�1

sodium polyphosphate in PBS. Samples (9 lL) were applied on glassslides coated with gelatin and dehydrated by passing them throughan ethanol series (50%, 80%, and 100%, v/v) for 3 min each and thenair-dried. Two times of hybridizations were performed at 46 �C for2 h each in a hybridization buffer [0.9 M NaCl, 40% formamide,20 mM Tris–HCl, 0.01% sodium dodecyl sulfate (SDS), pH 7.2]:Firstly, cells were stained with 4,6-diamidino-2-phenylindole(DAPI), as described by Juretschko et al. (1998), and secondly, the oli-gonucleotide probe S-�-Amx-0820-a-A-22 (Amx820, 50-AAAACCCCTCTACTTAGTGCCC-30, ordered from TaKaRa, Dalian, Chi-na) labeled with Cy5 was used for the hybridization of anammoxbacteria (Schmid et al., 2000). The Amx820 probe is specific foranammox genera Brocadia and Kuenenia (Schmid et al., 2005) andcan detect the most popular anammox bacteria that had a high de-gree of relatedness with Candidatus ‘‘Brocadia anammoxidans’’ andCandidatus ‘‘Kuenenia stuttgartiensis’’. After hybridization, a wash-ing step was performed at 48 �C for 20 min in a washing buffer(56.3 mM NaCl, 0.01% SDS, 20 mM Tris–HCl, pH 7.2). Subsequently,slides were rinsed with deionized water. For image acquisitions,an epifluorescence microscope (OlympusBX51, Japan) equippedwith a BX2 U-MWU2 laser (330–385 nm) was used together withthe standard software package delivered with the instrument(version 4.0).

3. Results and discussion

3.1. Reactors performance

Washing-out of biomass was observed during R1 operation.During the operation of R2 and R3, 100 mL effluent was collectedand centrifuged at 3200�g for 30 min. No pellet was visually ob-served, indicating basically no sludge washout occurred. The EMBReffluent TSS was below 3.0 mg L�1 while the SBR effluent TSS ran-ged from 7.5 to 19.4 mg L�1. The nitrogen removal performance ofthe reactors (R1, R2 and R3) is shown in Fig. 2. At the same HRT(48 h) for each reactor the concentration of ammonium and nitritein synthetic wastewater was increased gradually (Fig. 2).

For R1 (SBR) operation can be divided into three obvious phases:unstable phase, stable phase and inhibition phase. Similar phaseswere reported by Tang et al. (2010b). Unstable phase occurred dur-ing the first three weeks after inoculation, which was caused by thenegative response of bacteria in the biomass to sudden change oftheir environment and feed composition. After inoculation, thegranular sludge inoculated in R1 disaggregated, the dead cells wereanaerobically degraded into organic carbon and ammonium (fromorganic nitrogen). This caused accumulation of ammonium in R1effluent and denitrification of nitrite as shown Fig. 2B. No anammoxactivity was observed during the unstable phase due to the compe-tition for nitrite between anammox bacteria and DNB and limited

Fig. 2. Nitrogen removal performance of the reactors R1, R2 and R3. R1 (SBR) wasmodified to be operated as R2 (EMBR) on day 219 (black line). (A) The performanceof R1 (day 1–218) and R2 (day 219–397). (B) The start-up period of R1 in detail. (C)R3 (EMBR) performance during day 1–242. HRT of all reactors was 48 h.

Table 2Anammox activity of biomass from R1, R2 and R3.

Time Anammox activity (mg-N g�1 VSS day�1)

R1 R2 R3

Start-upa 0.515 ± 0.030 1.219 ± 0.139 0.338 ± 0.047Endb 1.219 ± 0.139 15.075 ± 0.586 1.043 ± 0.090

a The ‘‘start-up’’ date of R1 and R3 was defined as the end of unstable phase (day21 and day 25, respectively).

b Samples of R1, R2 and R3 were taken on day 216, 393 and 196, respectively.

76 Y. Tao et al. / Bioresource Technology 104 (2012) 73–80

population in the inoculum. After day 20, the ammonium concen-tration in R1 effluent dropped below the influent one, indicatingthe end of degradation of dead cells in the inoculated granules. Afteradditional 10 days, the nitrogen loading rate (NLR) of R1 was in-creased from 0.03 to 0.13 kg-N m�3 day�1 (day 21–147). Theammonium and nitrite removal of R1 reached to average 83.7%and 92.5%, respectively, during the stable period. Inhibition phaseoccurred as NLR increased further with high nitrite concentration.After the nitrite concentration was further increased over 10 mM,the nitrite concentration greater than 10 mM appeared to be inhib-itory to the activity of anammox bacteria because both ammoniumand nitrite removal, dropped below 47% and 78%, i.e. reduced by25–40% due to nitrite inhibition (Bettazzi et al., 2010). Anammoxactivity was restored when the ammonium and nitrite load was re-duced afterwards. Similar observation was reported previously(Tang et al., 2009). After 218-day incubation, the color of biomassin R1 changed from brown to russet.

For R2 (EMBR) operation, no unstable phase was observed be-cause this reactor was modified directly after R1 operation aftersuccessful anammox enrichments in R1. Under the MBR operationmode with no sludge discharge and continuous feeding, higher ratewas achieved i.e. the NLR increased from 0.05 to 0.25 kg-N m�3 day�1, increased by almost twice of that of R1. Although ni-trite inhibition in R2 was also observed when nitrite concentrationwas higher than 17 mM but the removal of ammonium and nitritewas still above 78% and 94%, being reduced by only 5–10% due tonitrite inhibition. This suggested that the microbial community inEMBR had stronger tolerance to nitrite inhibition than SBR.

The R3 (EMBR) was started with anoxic sludge and experiencedthe similar operational phases to R1. The unstable phase lasted about25 days. Afterwards, the NLR was gradually increased from 0.04 to0.23 kg-N m�3 day�1. In the stable phase, the color of R3 sludgeturned from dark brown to light castaneous. For the inhibition phase,however, unlike the EMBR R2, the nitrite inhibition (day 197–242)was more severe than that observed in R3. As influent nitrite reachedover 14 mM, nearly 50% of ammonium removal capability was lost.

3.2. Characterization of biomass

To characterize the growth of anammox activity of biomass inthree reactors, the specific anammox activity of the biomass fromeach reactor was determined using batch test (Table 2). The results

indicated that the specific anammox activity of R1, R2 and R3 bio-mass was enhanced by 130%, 1130% and 208%, respectively, by theend of each test. The highest specific activity was observed for R2biomass, which was developed from biomass at the end of R1 testfor additional 178 days. If R3 were operated for longer period thespecific activity would be increased accordingly. The average dailyincrease in specific anammox activity were 3.2, 77.8, 3.6 ng-N g�1VSS day�1 in R1, R2 and R3, respectively. Based on the specificrates (Table 2), the daily relative increase of the activity (% per day)was 0.60, 6.38 and 1.06 in R1, R2 and R3, respectively, suggestingthat EMBR was likely better for enrichment of anammox bacteriausing biomass with anammox activity (in R2 case) than start-upof a new reactor with inoculum with low activity, and in either sit-uation MBR performed better than SBR.

No granules were formed in R1 (SBR) during 219 day opera-tional period. It may be due to a long the HRT (48 h) used for R1and long settling time (30 min). A short HRT (several hours or less)may help granulation of anammox sludge (Tsushima et al., 2007).Short settling time may provide strong selective pressure for gran-ulation (Graaff et al., 2010). In both EMBRs, biomass was in floccu-lent form throughout the tests.

3.3. FISH analysis

FISH analysis indicated the existence of anammox bacteria in theinoculum sludge of both R1 (laboratory aerobic granular sludge)and R3 (full-scale WWTP activated sludge) (Fig. 3A and D). Basedon the FISH data, the anammox bacteria in the three reactors wereeither Candidatus ‘‘Brocadia anammoxidans’’ or Candidatus ‘‘Kuene-nia stuttgartiensis’’. In this study, the initial anammox bacterialpopulations in the two inoculum sludge were different. Only quiteminor green fluorescence in WWTP sludge was observed (Fig. 3D),which indicated the presence of tiny amount of anammox bacteriain WWTP sludge for R3 inoculum. In contrast, the FISH imageshowed more broad distribution of anammox bacteria in R1 inocu-lum sludge. After operation for 218-day in R1 and 241 days in R3,FISH image suggested that aerobic granular sludge used for R1was better inoculum to start up an anammox reactor than the acti-vated sludge because the targeting bacteria covered much morearea in R1 than R3 (Fig. 3B, E and F). This observation was confirmedby the specific anammox activity analysis (Table 2). Interestingly,there was no significant increase in the fluorescence intensity ofanammox cells in R2 biomass than in R1 biomass although theanammox activity of the former was much higher than the latter(Table 2). This suggested that FISH technique is useful to identifythe presence of anammox bacteria qualitatively but cannot be usedto evaluate the activity quantitatively.

3.4. Microbial community structure

In this study, the changes in microbial community were moni-tored by using 16S rDNA-DGGE analyses. The DGGE profiles of bac-terial 16S amplicons obtained from microbial community DNAsisolated from R1 and R3, respectively, were compared at different

Fig. 3. FISH images of the R1, R2 and R3 biomass during the enrichment period. All bacteria (DAPI-strained) are displayed in blue. Anammox bacteria (Cy5-labeled Amx820,yellow) are displayed in green, as indicated by the color scheme in the panel below. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)

Y. Tao et al. / Bioresource Technology 104 (2012) 73–80 77

operational days (Fig. 4 and Tables 3 and 4). Similar trend of micro-bial community succession for both R1 and R3 were observed inDGGE data. Heterotrophic bacteria such as Rhodopseudomonas(band 7 in Fig. 4A) and Thermoanaerobacter pseudethanolicus (band1 in Fig. 4B) lost their dominance in both reactors over operationalperiod. Mixotrophic bacteria stayed much longer in R1 and R3 thanheterotrophic bacteria. For example, Desulfotomaculum acetoxidans(band 2 in Fig. 4B), an acetate utilizing sulfate reducing bacteria re-mained dominance until day 196. It took over 200 days to elimi-nate most non-anammox bacterial species in this study.

3.5. Side populations in EMBR and SBR

Since both EMBR and SBR were inoculated with mixed cultures,the change of side populations that related to nitrogen cycle wasalso comprehensively investigated in order to understand the rela-tionship between these bacteria and anammox bacteria in thereactor system. Many environmental factors, such as temperature,influent substrate or HRT, do not only influence the growth ofanammox bacteria but also the growth of side population (Huet al., 2010). In this scenario, the retaining of the side populationsin anammox reactors could be inevitable because of complexity ofthe microbial communities of inoculum sources (Fig. 4).

Batch experiments were conducted to determine the activitiesof the side populations involved in nitrogen cycle i.e. DNRA, AOB,NOB and DNB before and after anammox operation (Table 5). Theincrease in DNRA activity was found in both R1 and R3. DNRA pro-cess was widely discovered in nature (Lam et al., 2009; Ward et al.,2009), in which NHþ4 is originated from either NO�3 or NO�2 . Forreactor R3, DNRA bacteria were observed in 16S rDNA-DGGE image(Fig. 4, Table 4). DNRA activity was also determined by batch testsusing biomass from R2 and R3 (Fig. 5 and Table 5). The inoculumsludge of R3 (WWTP sludge) contained more DNRA-responsiblestrains than that of R1. Most importantly, after operation for242 days DNRA species still survived and maintained their activity.

This observation has not been described in other reports of anam-mox bioreactors. Further research is needed to understand the roleof DNRA bacteria in anammox process although these organismsare not desired for anammox actions.

The results of batch tests indicated that the activities of AOB,NOB and DNB were significantly decreased but these functionalgroups still survived in the biomass, which was in the same orderof magnitude as anammox bacteria in R1 and R3 but much lowerthan that in R3. Interestingly, any negative impact caused by theseside populations was not observed. The presence of AOB, NOB, DNBand DNRA bacteria, from an ecological perspective, could play a po-sitive role in the consumption of residual DO in influent andmetabolize the organic matter that generated after cell lysis.

3.6. MBR for anammox process

Batch test results showed anammox activity was increased by1130% and 208%, respectively, for R2 and R3 biomass, comparedwith 130% of R1 sludge (from SBR) (Table 1). This suggested thatMBR was more suitable for starting up anammox process thanSBR for enriching anammox bacteria.

An apparent advantage of MBR is its strong biomass retentioncapacity with little concern about sludge flotation or wash-outeven when the reactor is operated under high loading rate or shortHRT. Because anammox bacteria grow slowly, the high sludgeretention could maintain anammox bacteria at maximum in reac-tor system compared with SBR and other reactor in which floata-tion and washout of anammox biomass could happen as reportedin previous study (Ni et al., 2010b). For example, it was reportedthat washout of anammox biomass from an EGSB caused the de-crease of volumetric nitrogen removal rate from 4.00 to 2.46 kg-N m�3 day�1 (Chen et al., 2010).

Anammox bacteria belong to the category of slow-growingmicroorganism, and are still uncultivated in vitro. Based on ‘‘thegreat plate count anomaly’’ theory (Lam et al., 2009), slow-growing

Fig. 4. 16S rDNA-based DGGE profiles of the biomass from R1 (A) and R3 (B) at different days.

Table 3Name and similarity of each band from R1 biomass DGGE results.

Band No. Name of strains Similarity (%) Function related to nitrogen removal

1 Eubacterium 992 Yard-trimming-compost bacterium 963 Aminobacter sp. 1004 Geobacter sp. 926 Lachnospiraceae 987 Rhodopseudomonas 998 Gemmatimonadetes 9311 Petrimonas sp. 10014 Acinetobacter sp. 10016 Bosea sp. GSM-187 98 Denitrification17 Eubacterium WCHB1-40 9619 Deinococcus sp. 9620 Pseudoxanthomonas taiwanensis 9921 Nitrospira sp. 98 Nitrite oxidation22 Gemmatimonadetes 9723 Sphingobacteriales crenotrichaceae 96 Ammonium oxidation24 Nitrosospira sp. 95 Nitrite oxidation26 Planctomycetes 98 Anammox27 Nitrospira sp. clone Ss-05-20 100 Nitrite oxidation

Table 4Name and similarity of each band from R3 biomass DGGE results.

Band No. Name of strains Similarity (%) Function related to nitrogen removal

1 Thermoanaerobacter pseudethanolicus 992 Desulfotomaculum acetoxidans 913 Candidatus Accumulibacter phosphatis 994 Acidaminococcus fermentans 1005 Syntrophobacter fumaroxidans 996 Nitrosomonas eutropha 99 Nitrite oxidation7 Thermanaerovibrio acidaminovorans 1008 Planctomycetes 98 Anammox9 Ammonifex degensii 86 DNRA

78 Y. Tao et al. / Bioresource Technology 104 (2012) 73–80

microorganisms are capable of growth only at low nutrientconcentrations. This is a dilemma when to enrich such bacteria(Connon and Giovannoni, 2002). MBR, however, allows the slow-

growing cells to accumulate to extremely high density since almostall the biomass can be retained. To date, it has been reported that aMBR for culturing high purity anammox biomass had a volumetric

Table 5Specific nitrogen removal activity of biomass from R1 and R3.

Nitrogen removal activity (mg-N g�1 VSS day�1)

DNRA Denitrification Ammonia oxidation Nitrite oxidation

R1Start-upa 0.790 ± 0.575 212.751 ± 9.572 329.862 ± 17.853 486.908 ± 8.257Endb 1.867 ± 0.666 1.193 ± 1.486 7.815 ± 0.224 13.330 ± 1.175

R3Start-upa 1.350 ± 0.327 478.552 ± 14.933 132.873 ± 7.786 253.865 ± 15.441Endb 2.327 ± 0.477 1.630 ± 0.913 2.205 ± 0.617 2.690 ± 1.578

a The ‘‘start-up’’ date of R1 and R3 was defined as the end of unstable phase (day 21 and day 25, respectively).b Samples of R1, R2 and R3 were taken on day 216, 393 and 196, respectively.

Fig. 5. DNRA Capacity of the biomass from R2 and R3. Both R2 and R3 biomass were harvested at the end of the tests. (A) Nitrite as sole substrate (biomass of R2); (B) nitrite assole substrate (biomass of R3); (C) nitrate as sole substrate (biomass of R2); (D) nitrate as sole substrate (biomass of R3).

Y. Tao et al. / Bioresource Technology 104 (2012) 73–80 79

nitrogen loading rate below or equal to 120 mM-N L�1 day�1 withpurity of anammox cells over 97% (van der Star et al., 2008). Thisrate was much lower than other anammox bioreactor for the treat-ment of ammonium rich wastewater, some of which could reachnitrogen loading rate over 2000 mM-N L�1 day�1 (Hu et al.,2010). In this study, the loading rate of 0.25 kg-N m�3 day�1 or18 mM-N L�1 day�1 with biomass concentration of 1.2–1.5 g L�1

were achieved. The loading rate could be further enhanced byaccumulation of more anammox cell concentrations.

3.7. Inoculum source

Inoculum sludge is also an important factor for successful start-up of anammox reactor. Besides anammox biomass (Joss et al.,2009), anaerobic methanogenic granules (Tang et al., 2009,2010b), aerobic granular sludge (Ni et al., 2010a,b), nitrifying bac-terial culture (van der Star et al., 2007) and denitrifying bacterialculture (Tsushima et al., 2007) from laboratory or WWTPs werealso reported as inocula of anammox reactors. In this study, twodifferent inoculum sources for R1 and R3 were used, respectively.FISH images (Fig. 3A and D) indicated that both inocula containedanammox bacteria but the WWTP sludge contained less fluores-cence intensity of anammox bacteria than aerobic granular sludge.It was likely the granular sludge had more anammox bacteria thanflocculent sludge because the granular structure would protectanaerobic bacteria better than flocculent sludge. Due to slowgrowth rate of anammox bacteria, it is advisable to select inoculumwith relatively high concentration of anammox bacteria.

4. Conclusions

A better performance in starting up anammox process by MBR,compared to SBR, was demonstrated in this study. Modification ofa SBR as MBR with installation of an external membrane moduleresulted in acceleration of specific anammox activity by 19 times.16S rDNA-DGGE analyses demonstrated that the majority of sidemicrobial populations were eliminated, but still a part was re-tained. No negative impact caused by these side populations wasobserved. The start-up with MBR appeared to be more effectivethan SBR for the enrichment of anammox bacteria due to highsludge retention property of MBR configuration.

Acknowledgements

This research was supported by National Natural ScienceFoundation of China (No. 21177033), the Research Fund for theDoctoral Program of Higher Education, Ministry of Education ofPR China (20092302110059), the National Key Technology R&DProgram (2009BADC2B01-04-02), the Heilongjiang Natural ScienceFund for Distinguished Young Scientists (JC200909), and the StateKey Laboratory of Urban Water Resource and Environment, HarbinInstitute of Technology (QAK201016, 2010DX04).

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