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ARTICLE Impact of oxygen on the coexistence of nitrification, denitrification, and sulfate reduction in oxygen-based membrane aerated biofilm Hong Liu, Shuying Tan, Zhiya Sheng, Tong Yu, and Yang Liu

Abstract: Membrane aerated biofilms (MABs) are subject to “counter diffusion” of oxygen and substrates. In a membrane aerated biofilm reactor, gases (e.g., oxygen) diffuse through the membrane into the MAB, and liquid substrates pass from the bulk liquid into the MAB. This behavior can result in a unique biofilm structure in terms of microbial composition, distribution, and community activity in the MAB. Previous studies have shown simultaneous aerobic oxidation, nitrification, and denitrification within a single MAB. Using molecular techniques, we investigated the growth of sulfate-reducing bacteria (SRB) in the oxygen-based MAB attached to a flat sheet membrane. Denaturing gradient gel electrophoresis of the amplified 16S rRNA gene fragments and functional gene fragments specific for ammonia-oxidizing bacteria (amoA), denitrifying bacteria (nirK), and SRB (dsrB) demonstrated the coexistence of nitrifiers, denitrifiers, and SRB communities within a single MAB. The functional diversities of SRB and denitrifiers decreased with an increase in the oxygen concentration in the bulk water of the reactor. Key words: membrane aerated biofilms, sulfate-reducing bacteria, functional diversity, PCR–DGGE. Résumé : Les biofilms aérés a` membrane (MAB) sont sujets a` une « contre-diffusion » de l’oxygène et des substrats. Dans un réacteur a` biofilm aéré a` membrane, les gaz tel l’oxygène se diffusent a` travers la membrane jusque dans le MAB et les substrats liquides passent de la masse liquide au MAB. Ce phénomène donne naissance a` une structure de biofilm unique en son genre quant a` la composition, la distribution et l’activité des communautés microbienne du MAB. Des études antérieures ont révélé des activités aérobies d’oxydation, de nitrification et de dénitrification simultanément dans un même MAB. Au moyen de techniques moléculaires, nous avons examiné la croissance de bactéries sulfatoréductrices (SRB) dans des MAB oxygénés attachées a` un feuillet membranaire plat. L’électrophorèse sur gel en gradient dénaturant de fragments amplifiés du gène de l’ARNr 16S et de fragments de gènes fonctionnels liés uniquement aux bactéries oxydant l’ammoniac (amoA), aux bactéries dénitrifiantes (nirK) ou aux SRB (dsrB) a démontré la coexistence de communautés de nitrifiants, de dénitrifiants et de BRS dans un seul et même MAB. Les diversités fonctionnelles des SRB et des dénitrifiants ont subi une baisse en réponse a` la hausse de la concentration d’oxygène dans le volume d’eau du réacteur. [Traduit par la Rédaction] Mots-clés : biofilms aérés a` membrane, bactéries sulfatoréductrices, diversité fonctionnelle, PCR–DGGE.

Introduction Membrane aerated biofilm reactors (MABRs) have much higher gas transfer efficiency and can be run at lower cost than conventional biofilm reactors (Cote et al. 1988; Brindle and Stephenson 1996). In conventional biofilm reactors, aerobic and anaerobic processes are typically carried out in different vessels because the occurrence of nitrification and denitrification prevails in the presence and absence of oxygen, respectively (Zhao et al. 1999). In a MABR, oxygen and nutrients are provided to the biofilm from opposite directions; therefore, through manipulation of reactor conditions, simultaneous aerobic and anaerobic processes can be realized in a single biofilm cultured in one vessel (Syron and Casey 2008). A very different stratification in terms of substrate concentration and bacterial activity can be established within a membrane aerated biofilm (MAB) compared with a biofilm attached to a solid surface (Essila et al. 2000). In the development of MABRs to treat wastewater, it is important to understand the microbial processes and their stratification within the biofilm. It was first reported in 1988 that nitrification, aerobic oxidation, and denitrification simultaneously occurred in a single MAB

(Timberlake et al. 1988). Most studies and applications of MABRs in wastewater treatment have focused on organic carbon removal (Brindle et al. 1999), simultaneous nitrification and denitrification for nitrogen removal (Hibiya et al. 2003; Terada et al. 2003), and simultaneous chemical oxygen demand (COD) and nitrogen removal (Yamagiwa et al. 1994; Brindle et al. 1998; Semmens et al. 2003). The stratifications of nitrifiers and denitrifiers in terms of their activities and community structures have also been investigated (Schramm et al. 2000; Cole et al. 2002, 2004; Satoh et al. 2004; Matsumoto et al. 2007; Gong et al. 2008). Gilmore et al. (2013) demonstrated the coexistence of partial nitrification and anaerobic ammonium oxidation (anammox) for nitrogen removal. They found that aerobic bacteria (AOB) existed near the membrane side while the anaerobic denitrifiers were present in the anoxic environment near the surface of the biofilm. The expectation that anoxic strata of MABs provide a good environment for the growth of sulfate-reducing bacteria (SRB) is based on the observation of sulfate reduction in the deeper anoxic zone of conventional aerobic wastewater biofilm (Santegoeds et al. 1998; Okabe et al. 1999; Ito et al. 2002). Unfortunately, H2S production during the sulfate-reducing process promotes corrosion

Received 27 August 2014. Revision received 6 December 2014. Accepted 18 December 2014. H. Liu, S. Tan, Z. Sheng, T. Yu, and Y. Liu. Department of Civil and Environmental Engineering, Faculty of Engineering, University of Alberta, Edmonton, AB T6G 2W2, Canada. Corresponding authors: Yang Liu (e-mail: [email protected]) and Tong Yu (e-mail: [email protected]). Can. J. Microbiol. 61: 237–242 (2015) dx.doi.org/10.1139/cjm-2014-0574

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Fig. 1. Schematic drawing of a membrane aerated biofilm reactor (MABR).

of the facilities (Pol et al. 1998) and leads to an increase in oxygen consumption due to the internal re-oxidation of H2S (Okabe et al. 1998); these reactions may further result in enhanced energy consumption and may reduce the wastewater treatment efficiency of an MABR. To the authors’ knowledge, a study of SRB in an oxygen-based MABR has been reported only recently by Tan et al. (2014). Simultaneous multiple microbial processes in a piece of MAB, including aerobic oxidation, nitrification, denitrification, and sulfate reduction, were observed by microsensor measurements of O2, pH, oxidation–reduction potential (ORP), NH4+, NO3–, and H2S. However, Tan et al.’s study did not investigate the presence of nitrifiers, denitrifiers, and SRB in the biofilm. The present study evaluates the presence of nitrifiers, denitrifiers, and SRB and investigates the impact of oxygen on the functional diversity of these bacteria. Molecular biology methods currently applied in complex environmental samples (Sanz and Kochling 2007) were performed in our study. Within a single flat-sheet MAB, the functional genes encoding enzymes required for denitrification (nirK), ammonia oxidation (amoA), and sulfate reduction (dsrB) have previously been used as biomarkers for detection of denitrifying bacteria (Throback et al. 2004), ammonia-oxidizing bacteria (Nicolaisen and Ramsing 2002), and SRB (Geets et al. 2006), respectively.

Materials and methods Reactor design and operation A schematic drawing of the MABR used in the study is shown in Fig. 1. Biofilm was grown on a 25-␮m-thick silicon flat sheet (30 cm × 3 cm) microporous membrane (pore size: 0.12 ␮m × 0.04, polypropylene; Celgard, North Carolina, USA) installed in a reactor designed by Tan et al. (2010). The MABR was operated in a continuous mode with a synthetic wastewater influent rate of 2 mL/min, an effluent recirculation rate of 200 mL/min, and a hydraulic retention time of 5.6 h. The MABR was inoculated with activated sludge collected from the anaerobic digester at the Gold Bar Wastewater Treatment Plant in Edmonton. The influent synthetic wastewater contained 250 mg/L dextrose (COD), 5 mg/L KH2PO4, 20 mg/L NH4Cl, 277.5 mg/L Na2SO4, 12.86 mg/L MgCl2·6H2O, 2.57 mg/L FeSO4·7H2O, 0.26 mg/L CoCl2·6H2O, 0.77 mg/L CaCl2·2H2O, 0.26 mg/L CuSO4·H2O, 0.26 mg/L MnCl2·4H2O, 0.26 mg/L ZnSO4·7H2O, and 1 mg/L yeast extract. Pure oxygen was supplied from a cylinder (Cat. No. OX 2.6-T, Praxair, Canada) and the oxygen pressure was controlled at 50 kPa with a regulator (Cat. No. PRX 312-1331-540, Praxair). The oxygen flowing through the flat-sheet membrane was controlled with a flow meter (Ser. No. 066619, Cole Parmer). Experiments were conducted in 2 phases (P1 and P2). During P1, oxygen was supplied at

a flow rate of 20 mL/min; the dissolved oxygen (DO) concentration detected in the bulk water was 2.2 mg/L. The biofilm was drenched with bulk water (DO concentration 2.2 ± 0.28 mg/L) for several months. During P2, oxygen was supplied at a flow rate of 10 mL/min; the bulk water DO concentration was reduced from 2.2 mg/L to 0.64 mg/L for the subsequent 20 days, and then remained at 0.61 ± 0.3 mg/L for 5 consecutive months of operation. Analytical methods The bulk water quality was monitored with respect to pH, DO, ORP, COD, ammonium ions (NH4+), and sulfate ions (SO42–). All measurements were conducted after the samples were filtered with 0.22 ␮m membrane filters. Values of DO, pH, and ORP in the bulk water were measured on a daily basis by a DO probe (model No. Orion 97-08, Thermo Electron Corporation, California), a pH probe (Cat. No. 13-620-108, Accumet, Fisher Scientific, California), and an ORP probe (Cat. No. 13-620-81, Accumet, Fisher Scientific), respectively. SO42– was monitored using a Dionex Ion Chromatograph 2000 system (ICS-2000, Dionex, California). NH4+ was monitored using an ammonia probe (Cat. No. 13-620-509, Accumet, Fisher Scientific). Biofilm sampling At the end of each phase of operation, at least 3 biofilm samples with 3 mm × 3 mm surface area were taken from different locations in the reactor without sacrificing the membrane. The collected biofilm samples were kept in PowerBead Tubes provided in the Power Soil DNA Extraction kit (Mo-Bio, Carlsbad, California), and DNA extraction was processed immediately. Microbial community analysis DNA was extracted using the Power Soil kit (Mo-Bio) following the protocol provided by the manufacturer. The primer pair GC341F and 518R was used to amplify 16S rRNA gene fragments (Muyzer et al. 1993); in addition, primers targeting the genes amoA, nirK, and dsrB were used to amplify gene fragments of ammonia oxidizing bacteria, denitrifiers, and SRB, respectively. Sizes of the amplified fragments and information regarding the primer sets are listed in Table 1. Amplification was performed in a 50 ␮L reaction system using an Authorized Thermal Cycler (Eppendorf, Hamburg, Germany). The reaction mixture consisted of 32.5 ␮L of water, 2 ␮L of DNA template (⬃35 ng), 1× PCR buffer, 1.5 mmol/L MgCl2, 1 ␮L of forward and 1 ␮L of reverse primer at 25 ␮mol/L (each), 0.2 mmol/L dNTP mix, 2.5% DMSO per reaction, and 1.25 U of Taq polymerase (Invitrogen, Carlsbad, California). PCR programs for Published by NRC Research Press

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Table 1. Primers used in this study. Target gene

Primer set

Sequence (5=-3=)

16S rRNA

GC-341 F 518 R DSR4 R DSR2060 F F1aCu R3Cu AmoA-1 F AmoA-2 R-TC

5=-Clamp 1-CCTACGGGAGGCAGCAG-3= 5=-ATTACCGCGGCTGCTGG-3= 5=-GTGTAGCAGTTACCGCA-3= 5=-Clamp 2-CAACATCGTYCACCAGGG-3= 5=-ATCATGGT(C/G)CTGCCGCG-3= 5=-Clamp 3-GCCTCGATCAG(A/G)TTGTGGTT-3= 5=-Clamp 4-GGGGTTTCTACTGGTGGT-3= 5=-CCCCTCTGCAAAGCCTTCTTC-3=

dsrB nirK amoA

Amplified fragment size

Reference

⬃200 bp

Muyzer et al. 1993

⬃350 bp

Geets et al. 2006

470 bp ⬃490 bp

Throback et al. 2004 Nicolaisen and Ramsing 2002

Note: Clamp 1: 5=-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3=; Clamp 2: 5=-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3=; Clamp 3: 5=-GGCGGCGCGCCGCCCGCCCCGCCCCCGTCGCCC-3=; Clamp 4: 5=-CGCCGCGCGGCGGGCGGGGCGGGGGC-3=.

16S rRNA gene amplification and functional gene amplification are provided in the attached supplementary material Table S11. Community patterns based on 16S rRNA, dsrB, nirK, and amoA genes were visualized with DGGE (gels were 16 cm × 16 cm × 1 mm). Electrophoresis was performed in TAE (20 mmol/L Tris, 10 mmol/L acetate, 0.5 mmol/L EDTA, pH 7.4) at 60 °C. The 16S rRNA gene was run on a 6.5% (m/v) polyacrylamide gel with a gradient of 30%–60% (100% denaturants in a mixture of 7 mol/L urea and 40% (v/v) formamide) at a constant voltage of 150 V for 8 h. The dsrB gene was run on a 7.5% (m/v) polyacrylamide gel with a denaturing gradient of 30%–60% at 100 V for 16 h. The nirK and amoA genes were run on a 7.5% (m/v) polyacrylamide gel with a denaturing gradient of 40%–70% at 180 V for 6 h. Following electrophoresis, the gel was stained for 20 min with 5 ␮L of ethidium bromide in 300 mL of TAE buffer before being photographed by UV transillumination (Viber Lourmat, France). For sequencing, migrated bands were excised from the polyacrylamide gel using a sterilized blade, and placed in 50 ␮L of Tris–HCl solution overnight to dissolve the DNA. A 2 ␮L volume of the solution was used as a DNA template and reamplified by PCR. PCR products for subsequent sequencing were purified using the Exosap-IT clean-up kit (Amersham Biosciences, Buckinghamshire, England), then the templates were diluted with laboratory-grade water to a concentration of 0.1–0.3 ng/␮L. In 1.5 mL tube, 10 ␮L of each sample was mixed with 1 ␮L of 3.2 pm/␮L reverse primer 518 R and sent to the The Applied Genomics Core center at the University of Alberta for Sanger sequencing. The obtained sequences were submitted to the GenBank nucleotide database, and BLAST (http://www.ncbi.nlm.nih.gov) was used to identify the most similar sequences in the database.

Results and discussion Reactor performance During P1, the pH was 7.6 ± 0.2 and the ORP was 67 ± 12 mV. The mean COD, NH4+, and SO42– removal efficiencies were 64%, 42%, and 27%, respectively. We observed biofilm sloughing at this stage, and a location near one corner of the membrane was exposed to the bulk water and was not covered with biofilm. In this stage, the surface anaerobic biomass may lose mass because of the higher oxygen in the bulk water. During P2, the mean pH and ORP were 7.6 ± 0.2 and –304 ± 34 mV, respectively. The mean COD, NH4+, and SO42– removal efficiencies were 73%, 45%, and 41%, respectively. The exposed zones of the membrane were covered with new biofilm growth, and the biofilm at the end of this stage was relatively evenly displayed on the membrane; the biofilm thickness was around 2000 ␮m. The removal efficiency of COD, NH4+, and SO42– and the chemical profiles detected by microsensors of NH4+, NO3–, and H2S within the stratified MAB suggested the simultaneous occurrence of nitrification, denitrification, and

1

sulfate reduction (Tan et al. 2014). The reactor operational data are shown in Fig. S11 in the supporting material. Microbial community analysis During P1, universal 16S rRNA gene primers were used to track SRB, denitrifiers, and nitrifiers within the MAB. 16S rRNA gene based DGGE fingerprints and phylogenetic analysis (Fig. 2) indicated that a diverse microbial community existed in the biofilm, as represented by the 23 bands displayed on the gel. Our detected sequences were close to those of known bacteria in Genbank, some of which have been shown to play functional roles in wastewater treatment plants. For example, Nitrosospira, Nitrosomonas, and Nitrospira (Daims et al. 2001; Wang et al. 2010) are responsible for nitrification (Gerardi 2006). The similarity of our sequences to those of Denitratisoma, Nitrosomonas, and Malonomonas (which is phylogenetically related to Desulfuromonales) (Kolb et al. 1998) may indicate the simultaneous presence of nitrifiers, denitrifiers, and SRB in our MAB sample. Owing to limitations in the PCR–DGGE technique, we failed to sequence 5 out of 28 bands. The complex environmental biofilm sample harbors a large diversity of species, and band separation might have been hampered (Miletto et al. 2007). Biases in PCR amplification of the 16S rRNA gene have been reported (von Wintzingerode et al. 1997). For example, in a DNA mixture, one set of primers may preferentially amplify the 16S rRNA gene of one species, while another primer pair preferentially amplifies the 16S rRNA gene of another species (Hansen et al. 1998). Therefore, further tests on dsrB, nirK, and amoA were performed. Functional gene analysis When DGGE fingerprints of dsrB, nirK, and amoA were obtained, more than 10 bands were distributed on the DGGE gel (Fig. 3). According to the BLAST results, the nucleotide sequence percent similarities between the detected genes and those in the database ranged from 85% to 96%. In the dsrB DGGE fingerprint (Fig. 3), sequences of band 1 to band 9 showed similarities to EF065002.1, AB061536.1, EU717109.1, AF273034, AF418194.1, FJ648437.1, EU426866.1, EF065059.1, and GQ324675.1, respectively. Bands 2 and 4 represented Desulfovibrio, and bands 5 and 6 sequences were similar to those of Desulfacinum and Desulfotomaculum, respectively. Principal wastewater treatment plant SRB Desulfovibrio and Desulfotomaculum were also detected in the MAB. Sequences of bands 1, 2, and 3 were similar to sequences of Roseobacter denitrificans (AJ224911), uncultured bacterium clone CYCU-0251 NirK-like (nirK) gene (DQ232408), and uncultured bacterium isolate DGGE gel band k41-4 nitrite reductase (nirK) gene (HM116369), respectively. Sequences of bands 1 and 2 of amoA were similar to those of uncultured Nitrosomonas (HQ541435 and GQ247383), and band 3 showed similarity to uncultured Nitrosospira (GU073249). Most of the other bands were

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-2014-0574. Published by NRC Research Press

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Fig. 2. Universal 16S rRNA gene primer (GC-341 F and 518 R) based DGGE fingerprints and phylogenetic tree based on 16S rRNA gene sequences of bacterial communities in an membrane aerated biofilm reactor (MABR). The scale bar represents 0.1 substitutions per nucleotide position. The tree is based on distance matrix analysis and a neighbor-joining method. Clustal X and Treeview software were used to do the analysis.

Fig. 3. Gene-specific primers based DGGE fingerprints: S1 and S2 represented different DNA samples taken from reactor during phase 2 (P2). The black triangles indicate bands that showed differences in terms of number or intensity.

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Liu et al.

similar to sequences of the uncultured bacterium isolate DGGE gel band ammonia monooxygenase-like (amoA) gene. More functional gene based DGGE bands appeared on the gel shown in Fig. 3 than were apparent in the 16S rRNA gene based DGGE pattern; that is, the functional gene primer set had much higher phylogenetic specificity than the 16S rRNA primer set. Previous studies compared the phylogeny derived from partial smallsubunit rRNA and functional gene sequences; it was demonstrated that the band pattern for functional gene amplicons was generally more complex than band patterns obtained by 16S rRNA gene amplicons (Joulian et al. 2001). However, this does not mean that fewer SRB, denitrifiers, and AOB species were detected by DGGE of 16S rRNA gene primer amplified products than detected by specific functional gene primer amplified products. One organism may carry more than one gene for a specific function, and different bacteria may carry identical functional genes with identical mobility on a DGGE gel (Nicolaisen and Ramsing 2002). Although functional gene based DGGE analysis could not provide detailed phylogenetic information such as that obtained with 16S rRNA gene based DGGE, it could provide information about functional diversity and physiological dynamics in complex multispecies biofilms. In the following experiment, functional gene based DGGE was applied to monitor the pattern changes under different oxygen concentrations in the bulk liquid. Impact of oxygen on functional diversity DGGE band patterns and relative intensities varied with changes in oxygen concentrations in the bulk liquid in contact with the biofilm. As shown in Fig. 3, when the detected DO concentration was high (P1), the DGGE bands were low in number and intensity, suggesting low SRB activity. When the bulk oxygen concentration was low (P2), the DGGE bands increased in number and intensity. Similar trends were observed for denitrifiers (nirK). For AOB band patterns (amoA), there were no obvious differences in band number and intensity when the biofilm was exposed to high and low bulk oxygen concentrations. The DGGE results showed that anaerobic bacteria (denitrifiers and SRB) were more sensitive than AOB to DO concentration in the bulk water. The presence and function of anaerobic bacteria, including denitrifiers and SRB, within the biofilm could be inhibited under higher bulk water DO condition. In addition, we observed sloughing of biofilm when the bulk water DO was at around 2 mg/L; the anaerobic microorganisms near the surface of the biofilm might have been sheared away, resulting in less functional diversity during phase 1. These factors both contributed to less functional diversity of denitrifiers and SRB during phase 1. Sulfate removal was less in P1 than in P2, which could be attributed to the suppression of sulfate reduction activity by the high DO in P1. It should be noted that before P1, the reactor had been running for over 1 year with almost zero oxygen in the bulk water. Although the reactor was running with a bulk water DO concentration of 2 mg/L for several months in P1, there were anaerobic denitrifiers and SRB present in the MAB. Therefore, a sample (sample 1) was taken at this point to see whether the anaerobic bacteria present in the MAB were reactivated, and sample 2 was taken several months later. We concluded from the DGGE bands present in phase 1 that some SRB and denitrifiers might have been reactivated; no substantial differences in the 2 samples in terms of band number and intensity were observed during P2, except that one more band (band 4) appeared on the dsrB gene gel in sample 2. As shown in Fig. S11, no significant difference in ammonium removal was observed when the bulk water DO was reduced from 2.2 mg/L (P1) to 0.64 mg/L (P2). This observation corresponds to our DGGE results, which showed that no obvious differences in DGGE band number and intensity were observed for AOB when the biofilm was exposed to different bulk water DO concentrations. Our results indicated that oxygen might not be the limiting factor for AOB. On the basis of previous assumptions of microorganism

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stratification within MABs (Schramm et al. 2000; Cole et al. 2002, 2004; Hibiya et al. 2003; Matsumoto et al. 2007), we concluded that nitrifying bacteria exist near the membrane side, while denitrifying bacteria and SRB prefer the anaerobic zone near the biofilm– bulk liquid interface. Thus, the anaerobic bacteria in the outer zone of the biofilm may form a protective barrier for AOB in the inner zone.

Conclusions DGGE analysis of the functional genes amoA, nirK, and dsrB confirmed the coexistence of AOB, denitrifiers, and SRB, respectively, in a single piece of MAB. The oxygen concentration in the bulk water had an impact on the microbial community behavior, particularly on anaerobic bacteria, including SRB and denitrifiers. The functional diversities of SRB and denitrifiers decreased with an increase of the oxygen concentration in the bulk water of the reactor. Our results suggest that sulfate reduction should be incorporated into the present MAB model that considers simultaneous organic carbon oxidation, nitrification, and denitrification. Future studies could examine how microbial community shifts can be related to reactor performance.

Acknowledgements The authors thank Sabinus Okafor and Miao Yu for operating the biofilm reactor, and acknowledge financial support from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canadian School of Energy and the Environment (CSEE), and the China Scholarship Council (CSC).

References Brindle, K., and Stephenson, T. 1996. The application of membrane biological reactors for the treatment of wastewaters. Biotechnol. Bioeng. 49(6): 601– 610. doi:10.1002/(SICI)1097-0290(19960320)49:63.0.CO;2-S. PMID:18626855. Brindle, K., Stephenson, T., and Semmens, M.J. 1998. Nitrification and oxygen utilisation in a membrane aeration bioreactor. J. Memb. Sci. 144(1–2): 197– 209. doi:10.1016/S0376-7388(98)00047-7. Brindle, K., Stephenson, T., and Semmens, M.J. 1999. Pilot-plant treatment of a high-strength brewery wastewater using a membrane-aeration bioreactor. Water Environ Res. 71(6): 1197–1204. doi:10.2175/106143096X122492. Cole, A.C., Shanahan, J.W., Semmens, M.J., and LaPara, T.M. 2002. Preliminary studies on the microbial community structure of membrane-aerated biofilms treating municipal wastewater. Desalination, 146(1–3): 421–426. doi:10. 1016/S0011-9164(02)00525-8. Cole, A.C., Semmens, M.J., and LaPara, T.M. 2004. Stratification of activity and bacterial community structure in biofilms grown on membranes transferring oxygen. Appl. Environ. Microbiol. 70(4): 1982–1989. doi:10.1128/AEM.70. 4.1982-1989.2004. PMID:15066788. Cote, P., Bersillon, J.L., Huyard, A., and Faup, G. 1988. Bubble-free aeration using membranes — process analysis. J. Water Pollut. Control Fed. 60(11): 1986– 1992. doi:10.2307/25046849. Daims, H., Nielsen, J.L., Nielsen, P.H., Schleifer, K.H., and Wagner, M. 2001. In situ characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67(11): 5273–5284. doi:10.1128/AEM.67.11.5273-5284.2001. PMID:11679356. Essila, N.J., Semmens, M.J., and Voller, V.R. 2000. Modeling biofilms on gaspermeable supports: concentration and activity profiles. J. Environ. Eng. ASCE, 126(3): 250–257. doi:10.1061/(ASCE)0733-9372(2000)126:3(250). Geets, J., Borrernans, B., Diels, L., Springael, D., Vangronsveld, J., van der Lelie, D., and Vanbroekhoven, K. 2006. DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria. J. Microbiol. Methods, 66(2): 194– 205. doi:10.1016/j.mimet.2005.11.002. PMID:16337704. Gerardi, M.H. 2006. Wastewater bacteria. John Wiley & Sons, Pennsylvania, USA. Gilmore, K.R., Terada, A., Smets, B.F., Love, N.G., and Garland, J.L. 2013. Autotrophic nitrogen removal in a membrane-aerated biofilm reactor under continuous aeration: a demonstration. Environ. Eng. Sci. 30(1): 38–45. doi:10. 1089/ees.2012.0222. Gong, Z., Liu, S.T., Yang, F.L., Bao, H., and Furukawa, K. 2008. Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal. Bioresour. Technol. 99(8): 2749–2756. doi:10.1016/j.biortech.2007.06.040. PMID:17702571. Hansen, M.C., Tolker-Nielsen, T., Givskov, M., and Molin, S. 1998. Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region. FEMS Microbiol. Ecol. 26(2): 141–149. doi:10.1111/j.1574-6941.1998. tb00500.x. Hibiya, K., Terada, A., Tsuneda, S., and Hirata, A. 2003. Simultaneous nitrificaPublished by NRC Research Press

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tion and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor. J. Biotechnol. 100(1): 23–32. doi:10.1016/S0168-1656(02)00227-4. PMID:12413783. Ito, T., Okabe, S., Satoh, H., and Watanabe, Y. 2002. Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions. Appl. Environ. Microbiol. 68(3): 1392–1402. doi:10.1128/AEM.68.3.1392-1402.2002. PMID:11872492. Joulian, C., Ramsing, N.B., and Ingvorsen, K. 2001. Congruent phylogenies of most common small-subunit rRNA and dissimilatory sulfite reductase gene sequences retrieved from estuarine sediments. Appl. Environ. Microbiol. 67(7): 3314–3318. doi:10.1128/AEM.67.7.3314-3318.2001. PMID:11425760. Kolb, S., Seeliger, S., Springer, N., Ludwig, W., and Schink, B. 1998. The fermenting bacterium Malonomonas rubra is phylogenetically related to sulfurreducing bacteria and contains a c-type cytochrome similar to those of sulfur and sulfate reducers. Syst. Appl. Microbiol. 21(3): 340–345. doi:10.1016/S07232020(98)80042-8. PMID:9841124. Matsumoto, S., Terada, A., Aoi, Y., Tsuneda, S., Alpkvist, E., Picioreanu, C., and van Loosdrecht, M.C.M. 2007. Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm. Water Sci. Technol. 55(8–9): 283–290. doi:10.2166/wst.2007.269. PMID:17546997. Miletto, M., Bodelier, P.L.E., and Laanbroek, H.J. 2007. Improved PCR–DGGE for high resolution diversity screening of complex sulfate-reducing prokaryotic communities in soils and sediments. J. Microbiol. Methods, 70(1): 103–111. doi:10.1016/j.mimet.2007.03.015. PMID:17481757. Muyzer, G., Dewaal, E.C., and Uitterlinden, A.G. 1993. Profiling of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reaction-amplified genes-coding for 16s ribosomal-RNA. Appl. Environ. Microbiol. 59(3): 695–700. PMID:7683183. Nicolaisen, M.H., and Ramsing, N.B. 2002. Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J. Microbiol. Methods, 50(2): 189–203. doi:10.1016/S0167-7012(02)00026-X. PMID:11997169. Okabe, S., Matsuda, T., Satoh, H., Itoh, T., and Watanabe, Y. 1998. Sulfate reduction and sulfide oxidation in aerobic mixed population biofilms. Water Sci. Technol. 37(4–5): 131–138. doi:10.1016/S0273-1223(98)00095-X. Okabe, S., Itoh, T., Satoh, H., and Watanabe, Y. 1999. Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms. Appl. Environ. Microbiol. 65(11): 5107–5116. PMID:10543829. Pol, L.W.H., Lens, P.N.L., Stams, A.J.M., and Lettinga, G. 1998. Anaerobic treatment of sulphate-rich wastewaters. Biodegradation, 9(3–4): 213–224. doi:10. 1023/A:1008307929134. PMID:10022065. Santegoeds, C.M., Ferdelman, T.G., Muyzer, G., and de Beer, D. 1998. Structural and functional dynamics of sulfate-reducing populations in bacterial biofilms. Appl. Environ. Microbiol. 64(10): 3731–3739. PMID:9758792. Sanz, J.L., and Kochling, T. 2007. Molecular biology techniques used in wastewater treatment: an overview. Process Biochem. 42(2): 119–133. doi:10.1016/j. procbio.2006.10.003.

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Satoh, H., Ono, H., Rulin, B., Kamo, J., Okabe, S., and Fukushi, K.I. 2004. Macroscale and microscale analyses of nitrification and denitrification in biofilms attached on membrane aerated biofilm reactors. Water Res. 38(6): 1633–1641. doi:10.1016/j.watres.2003.12.020. PMID:15016541. Schramm, A., De Beer, D., Gieseke, A., and Amann, R. 2000. Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm. Environ Microbiol. 2(6): 680–686. doi:10.1046/j.1462-2920.2000.00150.x. PMID: 11214800. Semmens, M.J., Dahm, K., Shanahan, J., and Christianson, A. 2003. Cod and nitrogen removal by biofilms growing on gas permeable membranes. Water Res. 37(18): 4343–4350. doi:10.1016/S0043-1354(03)00416-0. PMID:14511704. Syron, E., and Casey, E. 2008. Membrane-aerated biofilms for high rate biotreatment: performance appraisal, engineering principles, scale-up, and development requirements. Environ. Sci. Technol. 42(6): 1833–1844. doi:10.1021/ es0719428. PMID:18409602. Tan, S.Y., Yu, T., and Shi, H.C. 2010. Microsensor determination of sulphatereducing activity in an oxygen-based membrane-aerated biofilm. Conference Proceedings of IWA World Water Congress and Exhibition, 19–24 September 2010, Montréal, Que., Canada. Tan, S.Y., Yu, T., and Shi, H.C. 2014. Microsensor determination of multiple microbial processes in an oxygen-based membrane aerated biofilm. Water Sci. Technol. 69(5): 909–914. doi:10.2166/wst.2013.730. PMID:24622536. Terada, A., Hibiya, K., Nagai, J., Tsuneda, S., and Hirata, A. 2003. Nitrogen removal characteristics and biofilm analysis of a membrane-aerated biofilm reactor applicable to high-strength nitrogenous wastewater treatment. J. Biosci. Bioeng. 95(2): 170–178. doi:10.1016/S1389-1723(03)80124-X. PMID:16233387. Throback, I.N., Enwall, K., Jarvis, A., and Hallin, S. 2004. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol. Ecol. 49(3): 401–417. doi:10.1016/j.femsec. 2004.04.011. PMID:19712290. Timberlake, D.L., Strand, S.E., and Williamson, K.J. 1988. Combined aerobic heterotrophic oxidation, nitrification and denitrification in a permeablesupport biofilm. Water Res. 22(12): 1513–1517. doi:10.1016/0043-1354(88)90163-7. vonWintzingerode, F., Gobel, U.B., and Stackebrandt, E. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21(3): 213–229. doi:10.1111/j.1574-6976.1997. tb00351.x. PMID:9451814. Wang, X.H., Wen, X.H., Criddle, C., Wells, G., Zhang, J., and Zhao, Y. 2010. Community analysis of ammonia-oxidizing bacteria in activated sludge of 8 wastewater treatment systems. J. Environ. Sci. China. 22(4): 627–634. doi:10. 1016/S1001-0742(09)60155-8. PMID:20617742. Yamagiwa, K., Ohkawa, A., and Hirasa, O. 1994. Simultaneous organic-carbon removal and nitrification by biofilm formed on oxygen enrichment membrane. J. Chem. Eng. Jpn. 27(5): 638–643. doi:10.1252/jcej.27.638. Zhao, H.W., Mavinic, D.S., Oldham, W.K., and Koch, F.A. 1999. Controlling factors for simultaneous nitrification and denitrification in a 2-stage intermittent aeration process treating domestic sewage. Water Res. 33(4): 961–970. doi:10.1016/S0043-1354(98)00292-9.

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