Bioresource Technology 143 (2013) 178–186

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Bioresource Technology

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Denitrification of simulated municipal wastewater treatment planteffluent using a three-dimensional biofilm-electrode reactor: Operatingperformance and bacterial community

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.06.001

⇑ Corresponding authors. Address: College of Architecture and Civil Engineering,Beijing University of Technology, Beijing 100124, China. Tel.: +86 1067391726x8005; fax: +86 10 67391648 (R. Hao), tel.: +1 250 9606397; fax: +1250 9605845. (J. Li).

E-mail addresses: haoruixia@bjut.edu.cn (R. Hao), Jianbing.Li@unbc.ca (J. Li).

Ruixia Hao a,⇑, Sumei Li a, Jianbing Li b,⇑, Chengcheng Meng aa Key Laboratory of Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, Chinab Environmental Engineering Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada

h i g h l i g h t s

� A 3-D biofilm-electrode reactor was used for wastewater denitrification.� A nitrate removal of 98.3% was obtained with C/N ratio of 3.0 and HRT of 7 h.� Both heterotrophic and autotrophic bacteria were responsible for nitrate removal.� A phylogenetic tree of gene sequences in biofilm was established.� The biofilm was abundant with Thauera-like and Enterobacter-like bacteria.

a r t i c l e i n f o

Article history:Received 12 April 2013Received in revised form 30 May 2013Accepted 1 June 2013Available online 6 June 2013

Keywords:16S rDNABacterial communityBiofilm-electrode reactorCarbon sourceWastewater denitrification

a b s t r a c t

A three-dimensional biofilm-electrode reactor (3D-BER) was applied for nitrate removal from simulatedmunicipal wastewater treatment plant (WWTP) effluent. It was found that when the influent C/N ratioranged from 1.0 to 2.0, both heterotrophic and autotrophic denitrifying microorganisms played impor-tant roles in nitrate removal. The extension of hydraulic retention time (HRT) could enhance nitrateremoval, but too long HRT was not necessary. A phylogenetic tree of gene sequences in biofilm was estab-lished, and the biofilm was abundant with Thauera-like and Enterobacter-like bacteria. The results illus-trated that 3D-BER is a feasible and effective technology for the denitrification of WWTP effluent withpoor organic carbon source. A nitrate removal of 98.3% was obtained with C/N ratio of 3.0 and HRT of7 h. About 85.0–90.0% of nitrate removal was found at a C/N ratio of 1.5 and HRT of 10 h due to cooper-ative heterotrophic and autotrophic denitrification.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The shortage of water resources has become an exacerbating is-sue around the world as a result of accelerated industrializationand urban growth as well as changed climatic conditions. Amongvarious measures of addressing this challenging problem, waste-water reclamation has received tremendous attention (Yi et al.,2011). The reclaimed water from municipal wastewater treatmentplant (WWTP) can serve for many purposes, such as urban scenicand landscape greening, urban lake and river water replenishment,car washing, construction site water use, urban road sprinkling,industrial circulating cooling water use, irrigation water use, and

the recharge of groundwater (Hao et al., 2013). As a result, the re-claimed water represents an indispensable part of water resources.For example, the utilization of reclaimed water in Beijing in North-ern China (i.e. a region with serious water shortage problem)reached 0.65 and 0.68 billion m3 in 2009 and 2011, accountingfor about 18% and 20% of its total annual water consumption vol-ume, respectively (Wei et al., 2011). In general, the quality of re-claimed water is affected by the available treatment processeswhereas many pollutants could not be thoroughly removed. In par-ticular, WWTP effluent may still contain a relatively high concen-tration of inorganic nitrogen such as nitrate and nitrite. Forexample, a total nitrogen (TN) concentration (mostly nitrate) of15–40 mg/L in effluent was observed in many WWTPs in Beijing(Li et al., 2011). The discharge of nitrogen components into theenvironment through wastewater reclamation can lead to a num-ber of public health and environmental concerns. These includethe eutrophication of lakes and rivers, excessive vegetative growthand reduced fruit set of crops, decreased groundwater quality, and

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R. Hao et al. / Bioresource Technology 143 (2013) 178–186 179

other hazards (e.g., disease of methemoglobinemia in the fetus) forhuman and animal health (Ghafari et al., 2008). As a result, theeffective denitrification of WWTP effluent is of fundamentalimportance.

Biological denitrification has received the greatest interestsamong various processes since the bacteria can decompose nitrateinto harmless nitrogen gas with the presence of an electron donor(Karanasios et al., 2010). However, WWTP effluent usually containslow concentration of chemical oxygen demand (COD) with littleorganic carbon source or internal electron donor left for bacteriato utilize, and it is thus necessary to use an external source as elec-tron donor for denitrification. This problem can be addressed byusing a biofilm-electrode reactor (BER) which produces hydrogengas (H2) through water electrolysis, while the denitrifying microor-ganisms immobilized on the cathode surface directly contact thehydrogen gas and use it as an electron donor (Zhao et al., 2012).If the anode of BER is made of graphite, CO2 can also be generatedto serve as an inorganic carbon source for microorganisms and apH buffer in the denitrification system (Mousavi et al., 2012). Manystudies of using BERs have been reported. For example, Park et al.(2005) applied a BER with a dimensionally stable anode (DSA) anda graphite felt cathode to treat an aqueous solution containing highconcentrations of nitrate, and they observed a maximum nitrateremoval efficiency of 98% at an applied current of 200 mA, corre-sponding to a nitrate removal of 0.17 mg NO3�–N/(cm2 of biofilmsurface area day). Li et al. (2010) used Fe as a cathode and Ti/IrO2–Pt as an anode in an undivided cell to treat nitrate contami-nated wastewater, and they found that the nitrate concentrationdecreased from 100.0 to 7.2 mg/L within 180 min with the pres-ence of NaCl at 0.50 g/L, without the formation of ammonia and ni-trite by-products. Feng et al. (2013) and Huang et al. (2013) usedrectangular graphite electrodes in a cylindroid reactor to investi-gate the effects of different carbon sources and C/N ratio on thedenitrification efficiency, and they found that starch could be uti-lized by microbes to generate electrons, while the increase of C/Nratio from 2.0 to 3.5 resulted in an increase of nitrate removal from0.69 ± 0.02 to 1.09 ± 0.16 mg/(L h).

In general, the BER denitrification process can be affected bymany operating parameters, such as temperature, nitrate concen-tration, carbon source, salinity, pH, hydraulic retention time(HRT), electrode materials, reactor configuration, and currentintensity (Zhao et al., 2011). In conventional BER (with two elec-trodes), the cathode not only provides hydrogen for autotrophicdenitrifying bacteria as their electron donor, but also is used asthe carrier for microorganisms. As a result, its nitrate removalcapacity can be restricted by the surface area of cathode. Moreover,the denitrification can be affected by limited inorganic carbonsource in BER (Zhao et al., 2011). Consequently, the BER is usuallyassociated with a relatively low denitrification rate and requires along hydraulic retention time. To address this problem, a numberof efficient and economical alternatives of BER configuration havebeen proposed. These include the combined electrochemical reac-tor and biological reactor, and multi-electrode reactor or three-dimensional biofilm-electrode reactor (3D-BER) (Mousavi et al.,2012). In 3D-BER, activated carbon (AC) or AC mixed with anothersubstance is filled in the cathode part as a third bipolar electrode.This third electrode (i.e. AC filler) provides a high surface area forbiomass growth and attachment as well as increases hydrogengas yield. It also produces more CO2 to provide a desirable anoxiccondition where less organic carbon source is required to removenitrate (Zhou et al., 2007). For example, Zhou et al. (2009) usedPbO2 as an anode and activated carbon fiber (ACF) as a cathodeto treat nitrate-polluted groundwater, while the anode and cath-ode were set in the center and surrounding the inner wall of thereactor, respectively. The conventional BER (or 2D-BER) was con-verted to a 3D-BER when activated carbon (AC) was added and

packed in the reactor, and they found that the 3D-BER’s nitrate re-moval was increased from 57.93% to 78.10% as compared to 2D-BER at a HRT of 4 h, while the nitrate removal was further in-creased to 99.80% at a HRT of 8 h (Zhou et al., 2009). The 3D-BERwas also applied to oxidize pollutants from industrial solid wastelandfill leachate (Rao et al., 2009), remove acid orange 7 in simu-lated wastewater (Zhao et al., 2010), and remove NOx from fluegas (Zhou et al., 2012).

The novel 3D-BER technology is still in its early developmentstage with only a few denitrification studies being reported in lit-eratures, and even very few studies were found for nitrate re-moval from WWTP effluent. In addition, the denitrificationperformance of BER can be greatly affected by the involvedmicroorganisms. However, previous studies on both conventionalBER and 3D-BER technologies have mainly focused on reactor de-sign (e.g., electrode materials and reactor configurations) and theeffects of various operating factors. The information of involveddenitrifying bacterial community as well as the interaction be-tween bacteria community and reactor operating conditions arestill unclear (Mousavi et al., 2012; Feng et al., 2013). In fact, theunderstanding of microbial structure and diversity during denitri-fication process is of great importance for optimizing BER perfor-mance. The development of new molecular biology techniquessuch as the 16S rDNA-based methods and other methods likescanning electron microscopy (SEM) can make it possible to char-acterize and evaluate the bacterial community in BER (Park et al.,2006; Cho et al., 2008). Such techniques have been applied tocharacterize the microbial communities in many biological treat-ment studies of wastewater (Osaka et al., 2008; Srinandan et al.,2012). As described above, the 3D-BER can be a promising choicefor achieving a higher denitrification rate and a shorter HRT dueto the reactor’s unique configurations. It is thus of importanceto extend its application to WWTP effluent treatment. The objec-tive of this study was then to investigate the performance of a3D-BER for removing nitrate from simulated WWTP effluent un-der various C/N ratio and HRT conditions. The graphite cathodeand anode were used, and the activated carbon (AC) was filledin the reactor to form a third electrode. The bacterial communityin the biofilm developed on AC filler was characterized throughthe analysis of 16S rDNA. Such results can provide valuable infor-mation for designing more cost-effective denitrifying process forwastewater reclamation.

2. Methods

2.1. Experimental apparatus and simulated wastewater

Fig. 1 shows the schematic diagram of experimental setup andthe 3D-BER configuration. The reactor made of plexiglass wascylindrical with a diameter of 15 cm and a height of 56 cm. Thecathode consisting of six graphite rods (diameter of 2 cm andheight of 42 cm) was set surrounding the inner wall of the reactor,while the graphite rods were linked using insulated electrical cop-per wires. The anode was a graphite rod (diameter of 3 cm andheight of 42 cm) and was set in the center of the reactor. Theremaining space in the reactor was filled with granular activatedcarbon (AC) with particle size of 3–5 mm and a total volume of6 L. The reactor had an effective volume of 3.4 L. Prior to use, theAC was washed with H2SO4 solution (0.02 M) and deionized waterfor several times and was then dried at 105 �C for 24 h (Zhou et al.,2009). The AC filler can serve not only as a biofilm carrier but alsoas the third electrode. A DC regulated power supply (model HSPY-60-2 provided by Beijing Hansheng Puyuan Technology Co., Ltd. inChina, 0–60 V, 0–2 A) was used to provide constant current forwater electrolysis.

5

12 24

3

6

Fig. 1. Schematic diagram of 3D-BER apparatus (1) graphite rod anode (�1); (2)graphite rod cathode (�6); (3) activated carbon particles; (4) DC regulated powersupply; (5) peristaltic pump; 6 influent tank.

180 R. Hao et al. / Bioresource Technology 143 (2013) 178–186

Other reactor components consisted of an influent plastic tank(60 L) and a peristaltic pump. The influent water in this studywas a simulated WWTP effluent prepared by tap water amendedwith a stock solution of KNO3, CH3COONa, and phosphorus. Thenitrogen was provided by KNO3, with a NO3–N concentration of30 mg/L being maintained. The phosphorus was provided by KH2-PO4, with a PO43–P concentration of 0.3 mg/L being obtained. TheCH3COONa was added to provide carbon source according to de-sired C/N ratios, and no microelements were added since they wereavailable in tap water. About 20–30 L of synthetic wastewater wasprepared every 2 days, and its pH was adjusted to about 7.0 usingHCl and NaOH every morning on each day. Such simulated WWTPeffluent was used for the normal operation of 3D-BER after its bio-film development. Using a peristaltic pump, wastewater waspumped from the influent tank into the 3D-BER through its bottominlet, and flew through the biofilm on AC filler, and then flew outfrom the upper of the reactor.

2.2. Biofilm development and experimental procedure

The anaerobic sludge taken from an anaerobic–anoxic–oxic (A/A/O) process-based WWTP in Beijing was used to enrich the ni-trate-reducing microorganisms. The immobilization of denitrifyingbacteria to form biofilm on the surface of AC filler was preparedthrough adaptation, enrichment, immobilization and acclimatiza-tion (Zhou et al., 2009). About 10 L of seed sludge (i.e. a mixtureof wastewater and sludge) was added to a plastic tank (12 L). Anutrient solution of KNO3, CH3COONa, and KH2PO4 was added intothe tank according to C:N:P = 100:5:1, with a NO3�–N concentra-tion of 100 mg/L and pH of 7. By using a peristaltic pump, thesludge-water was driven to continuously circulate between the3D-BER and plastic tank through the reactor inlet and outlet. Nutri-ent solution was supplemented to the tank once everyday based ondesired C:N:P ratio. Such circulation was conducted for the first2 days until the yellowish-brown sludge was evenly distributedon AC filler.

After the first step, the immobilization and acclimatization ofbiofilm were conducted to enrich the denitrifying bacteria. Thetap water was amended with a stock solution of KNO3, CH3COONa,and KH2PO4 to form a NO3–N concentration of 155–178 mg/L and aCODCr concentration of 334–373 mg/L. Such nutrient-containingwater (about 40 L, amended once every 3 days) was added to the60-L influent tank, and its pH was adjusted once everyday to about7.0 with KH2PO4 and K2HPO4. The wastewater was pumped fromthe influent tank to flow through the 3D-BER, and was thendrained to the laboratory drainage system. To make microorgan-isms adapt to the environment, electric current in the 3D-BERwas gradually changing from 0 to 20, 20 to 40, and 40 to 60 mA,with each current level lasting for about 1 week. The influent andeffluent were run continuously, and the hydraulic retention time(HRT) was set at 7 h. This phase lasted for about 1 month, andthe effluent and influent concentration of nitrate were also moni-tored. After this phase, more than 90% of nitrate was steadily re-moved. A thick layer of dark-brown biofilm was visibly seen tocover the AC filler, and the effluent of 3D-BER was also seen withno suspended sludge, indicating that the biofilm had been wellformed. All bacterial inoculation and acclimation were carriedout during winter time at a room temperature of 10–15 �C.

After biofilm immobilization and acclimatization, the 3D-BERsystem was operated under different experimental conditions ofC/N ratio and HRT using simulated WWTP effluent as describedin Section 2.1. The simulated WWTP effluent was put in the 60-Linfluent tank, and was pumped to flow through the 3D-BER, andthen flew out of the reactor and was discharged to the laboratorydrainage system. A constant current of 40 mA was applied for allthe normal operation experiments. The C/N ratio was tested for0.5, 1.0, 1.5, 2.0, and 3.0, and the HRT was tested for 5, 7, 10, and12 h, respectively. The operational conditions (C/N and HRT) foreach run were maintained until steady nitrate removal for at least6 days. At every 24 h interval, 50 mL of sample was taken from thereactor effluent for nitrate and nitrite concentration analysis, andeach sample was taken for at least two successive HRTs.

2.3. Analytical methods

On a UV–Visible spectrophotometer (Shimadzu, Japan), the ni-trate (NO3�–N), nitrite (NO2�–N), and total nitrogen (TN) in watersamples were measured using ultraviolet spectrophotometry,spectrophotometry based on N-(1-naphthyl)ethylenediaminedihydrochloride, and ultraviolet spectrophotometry based on alka-line potassium persulfate digestion, respectively (SEPA, 2002).CODCr was measured using a COD analyzer (model 5B-6C providedby Lianhua Technology Co., Ltd., Beijing, China). pH was measuredusing a pH meter (model E-201-9 provided by Shanghai YokeInstrument Co., Ltd., China). The temperature of water samplewas measured using a thermometer that was fixed on the wall ofthe 3D-BER reactor. The removal of nitrate or total nitrogen (g)was calculated using the following formula:

g ¼ C0 � CtC0

� 100% ð1Þ

where C0 is the initial concentration of nitrate or total nitrogen (mg/L), and Ct (mg/L) is the remaining concentration of nitrate or totalnitrogen at time t.

2.4. Microbiological analysis

Bacterial community in the biofilm formed on the AC filler in3D-BER was analyzed by DNA-based molecular techniques. About20 mL of AC filler sample was taken from the 3D-BER after2 months of biofilm immobilization and acclimatization as wellas normal operation when its operating condition was C/N = 1.0,

R. Hao et al. / Bioresource Technology 143 (2013) 178–186 181

I = 40 mA, and HRT = 7 h. The sample was put in a 50-mL beaker tobe stirred by using a glass rod, and the biofilm was then strippedoff from the AC filler. The stripped biofilm sample (about 10 mL)was collected in a 10-mL centrifuge tube and stored at �80 �C be-fore analysis. The sample was sent to Sangon Biotech (Shanghai)Co., Ltd., in China (a professional biotechnology company) for thecharacterization of bacterial community by means of DNA extrac-tion, PCR amplification, cloning, sequencing and phylogeneticanalysis.

2.4.1. DNA extraction and PCR amplificationA detailed description of DNA extraction and PCR amplification

of 16S rDNA can be found in various literatures (Park et al., 2006;Brons and van Elsas, 2008). Total DNA was extracted from about0.2 mL of wet biofilm sample with the SK1201-UNIQ-10 column-type DNA extraction kit (Sangon Biotech Shanghai Co., Ltd., China)according to the manufacturer’s instructions. The DNA was precip-itated by adding ethanol before using as a template in polymerasechain reaction (PCR) amplification (Park et al., 2006). A forwardprimer (50f; 50-AACACATGCAAGTCGAACG-30) and a reverse primer(1492r; 50-GGTTACCTTGTTACGACTT-30) were used for PCR amplifi-cation. The PCR mixture contained 1 lL of extracted DNA, 10 lMconcentration of each primer (0.5 lL for each), 10 mM concentra-tions of deoxyribonucleotide triphosphate (dNTP) (0.5 lL), 2.5 lLof 10� Taq reaction buffer, and 5 U/lL concentration of Taq DNAPolymerase (0.2 lL) (Sangon biotech. Co., Ltd., Shanghai in China).The PCR mixture was added with sterile deionized water to obtaina final volume of 25 lL. The PCR amplifications were performed in0.2-mL reaction tubes on a model 2720 Thermal Cycler (BBI, Can-ada) using the following programs: initial denaturation for 5 minat 94 �C, 35 cycles of denaturation (30 s at 94 �C, 30 s at 55 �C,60 s at 72 �C), and a final extension of 72 �C for 8 min. All of thePCR amplification products were purified with the Agarose GelDNA purification kit (Sangon Biotech Shanghai Co., Ltd., China)according to the manufacturer’s instructions, and analyzed by elec-trophoresis in 1.0% (wt/vol) agarose gels followed by ethidium bro-mide staining (Brons and van Elsas, 2008).

2.4.2. 16S rDNA sequencing and phylogenetic analysisThe purified PCR amplicons were cloned using the T-carrier

cloning kit (Sangon Biotech Shanghai Co., Ltd., China) followingthe manufacturer’s instructions. After blue and white screening,102 positive (i.e. white) clones were randomly selected and thensequenced by a 3730 type DNA sequencing system (ABI, USA) usinga BigDye Terminator v1.1 sequencing kit (ABI, USA). The sequenceswere compared with those in the sequence database from Ribo-somal Database Project (RDP) (http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp). Sequences determined in this study and thoseretrieved from the database were aligned using a DNAStar SeqMansoftware (Brons and van Elsas, 2008). Phylogenetic tree was con-structed by a neighbor-joining algorithm using the MegAlign soft-ware (DNASTAR, USA) to obtain broader groupings to reveal thecompositions of bacterial community (Park et al., 2006).

2.4.3. Scanning electron microscopy (SEM) analysisThe surface morphology for microorganism in biofilm was

investigated by scanning electron microscopy (SEM). A series ofsample treatment procedures (e.g., fixation, washing, dehydration,drying, and coating) were applied before SEM analysis (Park et al.,2005; Cho et al., 2008). About 0.6 g of biofilm sample was put in a10-mL centrifuge tube (CT) and was centrifuged at 6000 rpm for5 min (model HC-2518 provided by Anhui Zhongke Zhongjia Scien-tific Instrument Co., Ltd., China). The supernatant was removed and7 mL of 2.5% glutaraldehyde was then added to mix with the set-tled sample in the bottom of CT. The CT was then placed in a refrig-erator at 4 �C for 4 h to allow for sample fixation. After that, the CT

was put in the centrifuge for centrifugation at 6000 rpm for 5 min,and the supernatant was removed after centrifugation. The left bio-film sample in the bottom of CT was added with about 7 mL ofultrapure water and was centrifuged again at 6000 rpm for5 min, and the supernatant was removed. Such washing throughcentrifugation was conducted for three times. The washed biofilmwas then dehydrated with 7 mL of 30%, 50%, 70%, 80%, and 90% eth-anol (static dehydration for 10 min for each concentration of etha-nol), respectively. The biofilm was finally dehydrated three timeswith about 7 mL of 100% ethanol (15 min for each time). At last,7 mL of ethanol and isoamyl acetate mixture (100% ethanol:iso-amyl acetate = 1:1) was poured into the CT staying for 15 min. Pureisoamyl acetate was then added for substitution for 15 min. Thesettled sample was put on a filter paper (10–15 lm) and was driedwith a vacuum dryer (Alpha 1–2 LD plus, CHRIST) for 24 h at a pre-cooling temperature of �10 �C. The dried sample was then sput-tered with a 10–20 nm gold layer (Puig et al., 2011). The coatedsample was examined with a SEM (HITACHIS-4300, Hitachi), andthe images were captured digitally.

3. Results and discussions

3.1. Effect of C/N ratio on denitrification performance

Fig. 2a and b present the variations of average nitrogen concen-tration and nitrogen removal under different C/N ratio conditionsduring the 3D-BER treatment process (with HRT = 7 h), while thenitrate (NO3–N) concentration in the influent was kept as a rela-tively constant value (i.e. 30 mg/L). The experiment for each C/Nratio was conducted for at least 6 days. It was observed that theC/N ratio greatly affected the accumulation of nitrite and the re-moval of nitrogen, with low C/N ratio beneficial for nitrite forma-tion. It was observed that the concentration of NO3–N in theeffluent of 3D-BER decreased from 16.69 to 12.15, 12.15 to 8.55,and 8.55 to 0.51 mg/L when the C/N ratio increased from 0.5 to1.0, 1.0 to 2.0, and 2.0 to 3.0, respectively. Similarly, the concentra-tion of nitrite (NO2–N) in the effluent decreased from 2.35 to 1.78,1.78 to 1.39, and 1.39 to 0.09 mg/L when the C/N ratio increasedfrom 0.5 to 1.0, 1.0 to 2.0, and 2.0 to 3.0, respectively. In general,denitrification efficiency increased with C/N ratio, and a high C/Nratio of 3.0 significantly enhanced the removal of nitrogen andthe restriction of nitrite accumulation. The removal efficiency of ni-trate increased from 48.9% to 62.2% when the C/N ratio increasedfrom 0.5 to 1.0, and a relatively stable nitrate removal efficiencyof around 71.7% was maintained for C/N ratio ranging from 1.5 to2.0. However, a very high nitrate removal of 98.3% was obtainedwhen the C/N ratio increased from 2.0 to 3.0. The removal effi-ciency of total nitrogen was lower than that of nitrate due to theformation of nitrite during the treatment process. The results ob-tained in this study were in agreement with results reported inprevious studies of BER treatment which indicated that high ni-trate removal efficiency (>95%) could be achieved when organiccarbon was abundant such as C/N > 2 (Zhao et al., 2012).

The denitrification effect can also be described using the ratio ofDC/DN, where DC and DN represent the difference of CODCr andtotal nitrogen (TN) between the 3D-BER influent and effluent,respectively. As a result, DC/DN ratio can be used to representthe approximate COD consumption per unit mass of nitrate re-moval. Fig. 2c presents the variation of DC/DN ratio under differentinfluent C/N conditions. The heterotrophic denitrification theoreti-cally requires 2.86 g of COD to remove 1 g of NO3–N, although theactual DC/DN ratio for complete nitrate removal is usually above3.0 due to the presence of oxygen and cell synthesis of microorgan-isms (Lee et al., 2001). In this study, sodium acetate (CH3COONa)was added into the simulated WWTP effluent as organic carbon

http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsphttp://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp

Fig. 2. Impact of C/N ratio on the denitrification effect of 3D-BER (experimentalcondition: flow rate = 504 mL/h; HRT = 7 h; T = 19 ± 2 �C; influent pH � 7.0;I = 40 mA).

Fig. 3. Impact of hydraulic retention time (HRT) on nitrogen removal in 3D-BER(experimental conditions: C/N = 1.5; T = 24 ± 2 �C; influent pH � 7.0; I = 40 mA.

182 R. Hao et al. / Bioresource Technology 143 (2013) 178–186

source for heterotrophic bacteria. It was observed from Fig. 2c thatDC/DN increased from 0.77 to 2.72 when the influent C/N in-creased from 0.5 to 3.0. All of these DC/DN ratios are below thetheoretical value of 2.86 for complete nitrate removal by heterotro-phic denitrifying microorganisms. This may indicate that both het-erotrophic and autotrophic denitrification microorganisms existedduring the 3D-BER treatment process. A high C/N ratio can acceler-ate the growth of heterotrophic denitrifying bacteria and thus pro-mote the total denitrification rate (Zhao et al., 2012). A study by Peiet al. (2010) found that a wetland based biological denitrificationremoved 70% of NO3–N at a C/N ratio of 7.0, but a complete re-moval of NO3–N and NO2–N was achieved at a C/N ratio of 8. In thisstudy, when the C/N ratio was 3.0, a DC/DN ratio of 2.72 wasachieved, and the nitrate removal and total nitrogen removalreached 98.3% and 98.0%, respectively, while the nitrite productionwas minimal. As a result, the heterotrophic denitrification mayhave played a major role at a relatively high C/N ratio when using

the 3D-BER process. However, when the organic carbon contentwas gradually decreasing in the reactor, the heterotrophic denitri-fying bacteria could be gradually domesticated into autotrophicdenitrifying bacteria, and the original autotrophic denitrifying bac-teria could gradually become the main microorganisms for nitrateremoval. The autotrophic microorganisms usually need a longadaptive phase with slow growth, and thus may lead to a lowerdenitrification rate (Zhao et al., 2011). For example, when the C/N ratio was 0.5, a DC/DN ratio of 0.77 was achieved, and the nitrateremoval of only 48.9% was obtained, with a considerable amount ofnitrite being accumulated. Thus, the autotrophic microorganismsmay have played a major role at a very low C/N ratio (i.e. 0.5).When the influent C/N ratio ranged from 1.0 to 2.0, both heterotro-phic and autotrophic denitrification microorganisms may playimportant roles in nitrate removal when using the 3D-BER, witha less considerable amount of nitrite being generated (Zhao et al.,2012). For example, when the C/N ratio was 1.0, 1.5, and 2.0, aDC/DN ratio of 0.99, 1.49, and 2.21 was achieved, and the averagenitrate removal of 62.2%, 70.4%, and 72.9% was obtained, respec-tively. The nitrate removal was nearly stable especially for C/N ra-tio of 1.5 and 2.0. Such cooperative heterotrophic and autotrophicdenitrification in 3D-BER system can lead to significant advantages(e.g., high nitrogen removal efficiency and thus larger treatmentcapacity) as compared with only autotrophic denitrification (Zhaoet al., 2011).

R. Hao et al. / Bioresource Technology 143 (2013) 178–186 183

3.2. Effect of HRT on denitrification performance

It was found from Fig. 2a that an influent C/N ratio of 1.5 wasassociated with a relatively satisfactory denitrification perfor-mance (i.e. nitrate and total nitrogen removal of 70.4% and65.2%). This C/N ratio was then used to examine the effect ofHRT on the denitrification performance of 3D-BER, and Fig. 3 pre-sents the results. The experiment for each HRT was conducted forat least 8 days. It was observed that the nitrate and total nitrogenremoval reached 75.9% and 70.5% at a HRT of 7 h and a temper-ature of 24 ± 2 �C, respectively. However, the corresponding re-moval was 70.4% and 65.22% (Fig. 2a) at a HRT of 7 h and atemperature of 19 ± 2 �C, respectively. Such difference mightmainly be caused by temperature variation during differentexperimental period, with higher temperature promoting nitrateremoval. It was found from Fig. 3 that HRT could greatly affectthe removal of nitrogen and the accumulation of nitrite. At lowHRT condition such as HRT = 5 h, a relatively lower denitrificationrate (e.g., nitrate and total nitrogen removal of 66.0% and 54.6%)was achieved, and a NO3–N and NO2–N concentration of above10 and 3.5–4 mg/L was observed in the effluent of 3D-BER,respectively. The denitrification rate increased with HRT, but itreached a maximum value (e.g., average nitrate removal of88.4%, ranging from 85.0% to 90.0%) when the HRT was 10 h. Fur-ther increase of HRT beyond 10 h resulted in the decrease of deni-trification rate. Longer HRT (i.e. 10–12 h) also led to lessaccumulation of nitrite in the 3D-BER effluent (e.g., less than1 mg/L). In the environment with relatively poor organic carbonsource (i.e. C/N = 1.5), both heterotrophic and autotrophic bacte-rial activities may be important, but the autotrophic bacteria needmore time to degrade nitrogen due to their relatively slowgrowth. As a result, a longer HRT could facilitate the nitrogen re-moval by such denitrifying bacteria and result in a lower nitriteaccumulation. However, in the continuous running mode of 3D-BER, too long HRT under relatively poor organic carbon sourcecondition could result in the endogenous respiration phase of het-erotrophic bacteria (i.e. a phase when bacteria live with nutritionin their bacterial body) for a long time. This would result in thereduction of heterotrophic bacteria and thus the poor denitrifica-tion effect, such as the reduced nitrate and total nitrogen removalof 79.0% and 76.0% at HRT = 12 h as shown in Fig.3. Consequently,when using 3D-BER for treating WWTP effluent with poor organiccarbon source, the extension of HRT could enhance the nitrogenremoval and restrict the nitrite accumulation, but too long HRTis not necessary due to the cooperative heterotrophic and auto-trophic denitrification mechanisms.

3.3. Bacterial community in 3D-BER

The analysis of 16S rDNA gene clones was conducted to assessthe bacterial community in the biofilm of 3D-BER. A total of 102

Table 1Phylogenetic groups in the biofilm of 3D-BER.

Phylogenetic group Number of clones Percentage (%)

Proteobacteriaa-proteobacteria 1 0.98b-proteobacteria 63 61.77c-proteobacteria 31 30.39e-proteobacteria 2 1.96

FirmicutesClostridia 3 2.94Bacilli 1 0.98

ActinobacteriaActinobacteria 1 0.98

Total 102 100

clones were obtained and sequenced without dividing operationaltaxonomic units (OUTs). Table 1 lists the summarized phylogeneticgroups of all of the analyzed bacterial clones. It can be found thatthe 3D-BER biofilm was dominated by Proteobacteria phylum inwhich the b- and c-Proteobacteria were the most abundant,accounting for 61.77% and 30.39% of the total clones, respectively.Only a small percentage (4.90%) of the total clones were related toother Gram-positive phyla of Firmicutes and Actinobacteria whichwere usually detected in anaerobic sludge digester and methano-genic inoculums (Ndez et al., 2008).

Proteobacteria was found to be dominant in many sulfur-based chemolithotrophic denitrification bioreactor studies (Ndezet al., 2008). Park et al. (2006) reported that Proteobacteria andflavobacteria were the dominant bacteria in a conventional bio-film-electrode reactor where the b-Proteobacteria played keyroles in the denitrification performance of biofilm reactor. How-ever, the flavobacteria was not detected in the biofilm of 3D-BERin this study. It was reported that b-Proteobacteria bacteria tendto survive in the anaerobic environment utilizing organic nutri-ents, while some of them may also utilize hydrogen, ammonia,methane and volatile fatty acids (Xia et al., 2010). Thus, theanaerobic operating environment in 3D-BER may facilitate thesurvival of b-Proteobacteria in the biofilm (i.e. 61.77% of the totalclones). The b-Proteobacteria detected in the biofilm sample wasfurther subdivided into classes and genera as shown in Fig. 4. Asignificant microbial diversity in the biofilm was observed.Fig. 4a shows that 57.15% of the total clones related to b-Prote-obacteria were pertaining to the family Rhodocyclaceae class (i.e.the genera of Thauera, Sulfuritalea and Azospira). Among them,83.0% of the total clones belonged to genus Thauera, which havebeen reported in many denitrifying bioreactor studies (Osakaet al., 2008; Daniel et al., 2009). Mao, 2009 examined 8 Thauerabacteria (Thauera aminoaromatica, Thauera linaloolentis, Thaueraphenylacetica, Thauera terpenica, Thauera sp. DNT-1, Thauera sp.27, Thauera sp. 28, and Thauera sp. 63), and found that all ofthem had denitrification function. It was found from Fig. 4a that33.33% of the b-Proteobacteria consisted of the family class ofComamonadaceae (Acidovorax, Albidiferax, Aquincola, Giesbergeria,Hydrogenophaga, Pelomonas, Polaromonas, Simplicispira, unclassi-fied Comamonadaceae), which had been found in denitrifyingreactors with acetate as the organic carbon source for microor-ganism (Ginige et al., 2005; Osaka et al., 2008). In this study,the organic carbon source in the 3D-BER was supplemented withsodium acetate, and this would lead to a relatively high percent-age of Comamonadaceae bacteria in the biofilm. The Burkholderi-ales-like bacteria (Aquabacterium, Ideonella, Rubrivivax,unclassified Burkholderiales) accounted for 9.52% of the b-Proteo-bacteria in the biofilm of 3D-BER, while this family class of bac-teria have been reported in the studies of anoxic/aerobicdigestion bioreactors (Zheng, 2012).

Unlike the prominent diversity of b-Proteobacteria in the 3D-BER biofilm, c-Proteobacteria contained three genera (Enterobacter,Citrobacter and Erwinia) (Fig. 4c), where the Enterobacter-like bac-teria of Enterobacteriaceae took up 88.89% of the clones belongingto c-Proteobacteria. Enterobacteriaceae-like bacteria were found tohave the physiological function of denitrification and polyphos-phate accumulation even under anoxic condition, although theirdenitrification capacity and aerobic phosphorus accumulationcapacity were excellent (Bao et al., 2007). This can explain the re-moval of both nitrate and phosphorous in 3D-BER. Fig. 5 presentsthe phylogenetic tree of gene sequences in 3D-BER biofilm, whereM represents a clone. For example, clone M89 represents the Enter-obacter-like bacteria. Such significant diversity of microbial com-munity in the biofilm as shown in Fig. 5 could enhance thedenitrification performance of 3D-BER for wastewater with lowC/N ratio.

Fig. 4. Distribution of b-and c-Proteobacteria in the biofilm of 3D-BER (a) percentage of b-proteobacteria classes; (b) percentage of genera in b-proteobacteria; (c) percentageof genera in c-proteobacteria (experimental condition: C/N = 1, HRT = 7 h, T = 19 ± 2 �C, I = 40 mA).

Fig. 5. Phylogenetic tree of gene sequences in 3D-BER (note: M represents a clone).

184 R. Hao et al. / Bioresource Technology 143 (2013) 178–186

R. Hao et al. / Bioresource Technology 143 (2013) 178–186 185

3.4. Surface morphology of biofilm in 3D-BER

The surface morphology and structure of biofilm in the 3D-BERunder different influent C/N ratio conditions were obtained (seeSupplementary Material). It was found that the microorganismsin the 3D-BER biofilm at relatively low C/N ratio (i.e. 1.0) weremainly short rod (1–2 lm) shaped bacteria, while those at higherC/N ratio consisted of both short rod (1–2 lm) and axiolitic shaped(0.5–1 lm) bacteria. This is in agreement with the results of 16SrDNA analysis which indicated that the biofilm was abundant withThauera-like and Enterobacter-like bacteria, while such bacteriawere mostly short rod shaped (Buchanan and Gibbone, 1984;Mao, 2009). It can also be found that the biofilm contained a muchless amount of bacteria at relatively low influent C/N ratio thanthat at a higher C/N ratio (see Supplementary Material). The reasonfor this can be explained that the growth of heterotrophic bacteriadepends on organic carbon source. The normal operation experi-ments of 3D-BER in this study started from a high C/N ratio (i.e.3.0) to a low C/N ratio (i.e. 0.5). The low C/N ratio could lead tothe slow growth of bacteria and thus decrease the amount and typeof heterotrophic bacteria (e.g., mainly short rod bacteria, 1–2 lm)as compared to environment condition with higher influent C/N(e.g., both short rod and axiolitic bacteria, 1–2 and 0.5–1 lm). Infact, it was visibly seen during the experiments that the C/N ratiodirectly affected the amount of biofilms, and the biofilms at C/N ra-tio of 0.5 and 1.0 were thinner and less intensive as compared tothose at a C/N ratio of 3.0. It was also found that an extensive porestructure existed in the biofilm, and this could facilitate the suffi-cient contact of bacteria with nitrogen components and the easyflow of nitrogen gas during the denitrification process, leading toenhanced denitrification rate. However, for the biofilm with lowC/N ratio, such pore structure was not obvious.

4. Conclusion

The 3D-BER technology was effectively used for removing ni-trate from simulated WWTP effluent. An average nitrate removalof 98.3% and 88.4% was obtained at C/N ratio of 3.0 and HRT of7 h as well as at C/N ratio of 1.5 and HRT of 10 h, respectively.The biofilm on the AC filler of reactor was dominated by Proteobac-teria in which the b- and c-Proteobacteria were the most abundant.The diverse and dynamic bacterial community composition duringtreatment could greatly improve the performance of 3D-BER andmake it an attractive denitrification technology for wastewaterwith poor organic carbon source.

Acknowledgements

This research was supported by the Natural Science Foundationof China (No. 51178005) and Beijing Natural Science Foundation(No. 8132017). The authors thank the journal editor and the anon-ymous reviewers for their comments and suggestions that helpedin improving the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.06.001.

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Denitrification of simulated municipal wastewater treatment plant effluent using a three-dimensional biofilm-electrode reactor: Operating performance and bacterial community1 Introduction2 Methods2.1 Experimental apparatus and simulated wastewater2.2 Biofilm development and experimental procedure2.3 Analytical methods2.4 Microbiological analysis2.4.1 DNA extraction and PCR amplification2.4.2 16S rDNA sequencing and phylogenetic analysis2.4.3 Scanning electron microscopy (SEM) analysis

3 Results and discussions3.1 Effect of C/N ratio on denitrification performance3.2 Effect of HRT on denitrification performance3.3 Bacterial community in 3D-BER3.4 Surface morphology of biofilm in 3D-BER

4 ConclusionAcknowledgementsAppendix A Supplementary dataReferences