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1 The Korean Journal of Microbiology, Vol. 45, No. 2, June 2009, p Copyright 2009, The Microbiological Society of Korea ³ w» y³ p š w zy œw w š œ y³ w w w ³. w» w y³ w yw.» y y³ w e ƒ w. ƒ w» w y y w sw, Palo Alto, Nine Springs, Marshall w y» l y³ ammonia monooxygenase subunit A clone library w. w» Nitrosomonas europaea, N. oligotropha, N.-like, Nitrosospira lineage w y³, N. communis, N. marina, N. cryotolerans lineage w y³ w. y³ w, sw, Palo Alto, Marshall w N. oligotropha lineage w y³ ƒ š, Nine Springs w N. europaea lineage w y³. wr, y³» (HRT, SRT, MLSS) y (, ph, COD, NH 3, NO 3- ) m Redundancy Analysis w w., COD NO 3 - ƒ w» y³ w m w w ùkû. Key words ý ammonia-oxidizing bacteria, ammonia monooxygenase subunit A (amoa), nitrification, redundancy analysis ƒ sw w y y ƒ w. w š, w s, w (20). ü sww ƒ sw w w s š š., y w ³ wš (1), w»» w š œ w wš. sw w» w, y w, w š ù, w w y š (11, 20). w y» ù ƒƒ y(nitrification) k (denitrification) w w (11). y» y³(ammoniaoxidizing bacteria) y³(nitrite-oxidizing bacteria) (NH 3 ) (NO 2 - ) (NO 3 - ) y w, k ³ y ƒ *To whom correspondence should be addressed. Tel: , Fax: heedeung@korea.ac.kr (N 2 ) y w. y y³ y³ w ƒ» y³ y w (20). y³ ³ yw š yk w s w w (22). w y Nitrosomonas europaea y³ w š š š ù(7), N. europaeaƒ y³ w ƒ w y» (7). w w» w x w w y³ y sw. Hiorns (4) w Nitrosospira spp.ƒ, Wagner (21) w Nitrosococcus mobilisƒ, Dionisi (3) w N. oligotrophaƒ w š š š. wr, Purkhold (15) Limpiyakorn (9) x w w y N. communis N. cryotolerans sww w y³ w š š. w» w y³ ¾ yw, w ¾ w r. p, y³ w» y k š. w» s» 112

2 Vol. 45, No. 2 y³ 113 Table 1. Wastewater treatment plants analyzed in this study and operational data for them WWTP Location Type of process Flow rate (m 3 /day) Solids retention time (days) Conc. of influent COD (mg /L) Conc. of influent nitrogen (mg TKN/L) Pohang Pohang, Korea Conventional activated sludge 80, Palo Alto Palo Alto, California, USA Trickling filter+nitrification 114, Marshall Marshall, Wisconsin, USA Aerated-anoxic Orbal 1, Nine Springs Madison, Wisconsin, USA Modified UCT 150, , ph» y,» w p y³ w ƒ. w ƒ w ù(8, 9, 13, 14) ƒ w w w l w, ƒ w r., w w» ƒ w. w w 4 ³ w» l y³ ammonia monooxygenase subunit A clone library w, ƒƒ clone m mw 7 lineage w. y³ s m w ù Redundancy Analysis (10) w w» w p w. ³ w» y sww (sw, w ), Palo Alto w (Palo Alto, Calif., USA), Nine Springs w (Madison, Wisc., USA), Marshall w (Marshall, Wisc., USA) ew 4 ³ y» l. sww y œ š. y w»ƒ ( )» ƒ (~20 o C) yƒ w š (Fig. 1). Palo Alto w y w». w 1 e e z. 65%» wƒ w» z y w» w. Palo Alto w k w œ e., w» w 2 e z ƒ e. Marshall w aeratedanoxic œ (13) y xk». w x w j e z, 1 e e š». ƒ Á y œ»œ œ w 0 mg/l g. y/k (simultaneous nitrification and denitrification) w y. w, y 0.5~5 mg/l wš. Nine Springs w University of Cape Town (UCT) œ š (13). w 1 e e z l y yw x». x» y y» w. 2 e e. UCT œ y» y ü xk, w y. ƒ w e,,, p Table 1 (13). yw sww , Palo Alto w , Nine Springs w , Marshall w ƒƒ y w. y³ y ƒ» s»» w grab sampling w z w. x ¼ z DNA ¾ -80 o C þ š w. NH 3 -N, NO 3 - -N, PO P d w» w»œ j»ƒ 0.2 µm w x w, BOD, COD, TKN, T-P w x. d w Standard Methods (2) w w. DNA, DNA s, cloning,» 1.5 ml y Tris-EDTA (ph 7.6) w z» y w, y 250 µl Tris-EDTA (ph 7.6) xk g. xk genomic DNA w, Soil Extraction kit (MoBio Laboratories,

3 114 Hee-Deung Park Kor. J. Microbiol USA) w. 20~50 ng genomic DNA s (Polymerase Chain Reaction: PCR) w. s y³ ammonia monooxygenase subunit A (amoa) t w, primer p amoa-1f (5 - GGGGTTTCTACTGGTGGT-3 ) amoa-2r (CCCCTCKGSAAA GCCTTCTTC-3 ; K G y T; S C G ùký) w (16). s PCR Core System I (Promega, USA) w. s DNA» e z, QIAEX II Gel Extraction kit (QIAGEN, USA) w w z cloning. Cloning pgem-t easy vector system (Promega) w. x y ³ l Wizard Plus Minipreps DNA Purification System (Promega) w plasmid DNA w. Insert» ABI Prism BigDye terminator (Applied Biosystems, USA) w. sw w, Nine Springs w, Marshall w y³ amoa» (12, 13), Palo Alto w y³ amoa» Stanford w y œw Chok Hang Yeung w. y³ ƒ w amoa» y³ amoa» l CLUSTAL X version 1.83 (19) w ƒ» distance matrix. vp l distance matrix w neighbor-joining m [Saitou and Nei method, (17)] w. vp w Bootstrap 100 (resampling trials) mw. m Tree Explorer version ( biol.metro-u.ac.jp/te/te_man.html) w x yw. m x y w amoa Purkhold (15) w 6 y³ lineage (, Nitrosomonas europaea, N. oligotropha, N. communis, N. marina, N. cryotolerans, Nitro sospira) m. m y³ w» y ³ w» w m Redundancy Analysis (RDA) w. RDA l (species) y x w š ƒ w (10). 3ƒ (, w», y³ lineage, y ) s wù t t w., t w» (point), y³ linage y y t t. t y m w w forward selection (18) w. RDA Canoco m vp (Plant Research International, Netherlands) w. wr, AOB w e y w Pearson correlation coefficient sƒw. Pearson correlation coefficient x» -1 1 tx. Excel 2007 vp (Microsoft, Korea) w. ƒ p y³» w» sww w y³ p ww. ƒ w k (total Kjeldahl nitrogen: TKN),,, Fig. 1 ùkü. ƒ TKN 29.7~35.4 mgn/l, 16.8~21.0 mgn/l ƒ j. w. sww y œ kw» w y k 17.4 mgn/l, 13.1 mgn/l d. Palo Alto w y w w ù( : 0.2 mgn/l), ƒ e d (23.5 mgn/l). Nine Springs w y w w ù[ : 0.08 mgn/l( ), 0.05 mgn/l( )], y» y ü» k z [ : 11.5 mgn/l( ), 14.6( )]. Marshall w Fig. 1. Influent and effluent nitrogen compounds for the six samples taken from four different wastewater treatment bioreactors.

4 Vol. 45, No. 2 y³ 115 Fig. 2. Neighbor-joining tree based on amoa gene sequences retrieved from this study (boldface type) and pure-culture AOB strains. Clone sequences exhibiting >97% identity are indicated by symbols in parentheses. Bootstrap values were determined based on 100 trials and shown at nodes greater than 50. y/k z ƒ ùkû. ƒƒ mgn/l d, ƒƒ mgn/l ùkû. y³ p sw, Palo Alto, Nine Springs, Marshall w l y w, ƒƒ w y³ amoa clone library w. sw l 8, Palo Alto l 31, Nine Springs ( ) l 24, Nine Springs ( ) l 16, Marshall ( ) l 15, Marshall ( ) l 18 amoa clone. amoa clone mw w» w amoa clone» GenBank ( l 42 y³ amoa» w mw w. Fig. 2 m N. oligotropha ( clone 49%), N. europaea (30%), N.-like (11%), Nitrosospira (9%) y³ swš. N. communis N. marina w clone. w, N. cryotolerans lineage w clone 1. N. communis, N. marina, N. cryotolerans lineage w y³ w» y w w y w w w. clone 11% w N.-like lineage Purkhold (15) w 6 lineage w mw ƒ cluster w w. ƒ y³ š (Fig. 3). sww N. oligotropha (50%)ƒ, Palo Alto w N. oligotropha

5 116 Hee-Deung Park Kor. J. Microbiol Fig. 3. Distribution of amoa clones based on seven different AOB lineages for the six samples taken from four different wastewater treatment bioreactors. (42%)ƒ, Nine Springs w N. europaea (58%)ƒ, Nine Springs N. europaea (81%)ƒ, Marshall w N. oligotropha (80%)ƒ, Marshall w N. oligotropha (83%)ƒ ƒ ƒ ƒ j w y³ lineage. w ƒƒ w» y w e. wr, Nine Springs Marshall w., Marshall w Nitrosospira lineage w y³ 20%ù, x. Nine Springs w N. europaea lineage w y³ 58%, 81% ƒw. y³ š,» y w yw. y³ y ƒ w y³» y m Redundancy Analysis w sƒw. 4 6 amoa clone N. europaea, N. oligo tropha, N.-like, Nitrosospira lineage 4 ù m w. N. communis, N. marina, N. cryotolerans lineage w amoa clone m g. y³ w» (CODin), k (TKNin), (NH 3 -Nin),» (Temp), w (HRT), šx (SRT), (MLSS),» (COD), (NH 3 -N), (NO - 3 -N) 11 ƒ. m w Fig. 4. RDA analysis of AOB lineages for the six different samples of activated sludge bioreactors and correlation with operational and environmental variables. The length of an arrow indicates the relative importance of the explanatory variables to the T-RF patterns, and the angle of an arrow indicates whether the explanatory variables increases or decreases in magnitude with respect to the indicated AOB lineage. (P<0.05)» COD NO 3 - -N, y³ w e ùkû (P>0.05). Fig. 4 RDA m ùkü, y³, w, y ƒ wù v t w. v,» COD N.-like Nitrosospira lineage (positive correlation) ƒ š. wr,» NO 3 - -N N. oligotropha, N.-like, Nitrosospira lineage (positive correlation) ƒ š ù, N. oligotropha N. europaea lineage ƒ. š w w» w w» N. oligotropha, N. europaea, N. communis, Nitrosospira lineage w y³ š (9, 15). (Fig. 2 and 3) š ew ƒ w., N. communis lineage w y³. w y³(n. communis Nm2 strain) Koops (5) w, m w š š. w, N. communis kw p y ww ¾ ³., Palo Alto, sw, Marshall w

6 Vol. 45, No. 2 y³ 117 N.-like lineage w y³. p, Palo Alto w 31 amoa clone 10 ƒ(32%) N.-like lineage w x, w y³ Palo Alto w w w w. Fig. 4, N.-like lineage» COD ƒ š. Palo Alto w» COD NO 3 - -N ƒƒ 55.8 mgn/l 23.5 mgn/l w w ƒ wš. ¾ N.-like lineage w y³, w y³ w p ³, y³ w COD w w ù y. Watson (22) š w y³» w ù š w., Nitrosococcus mobilis» (COD) w» w nitrite w š w, Nitrosospira tenuis formate glucoseƒ w š w. y³ ƒ» w. y³ y³ y³ w e. w, RDA ƒ y³ w m w, Fig. 4» ƒ ƒ š (Pearson correlation coefficient=0.86). w, ƒ y³ w e (6, 9), ƒ y³ w» ƒ w» y³ w e. ³ w» p, y³, y³» y w. w»,, p ³ w. y³ N. europaea, N. oligotropha, N.-like, Nitros ospira lineage w, w y p y³. m RDA w y³» y ùkü. w w, w» y y³ w,» p. w» œw s ww, Palo Alto w, Nine Springs w, š Marshall w Ì. š w p w w. š x wás :., q y». 2. APHA, AWWA, and WPCF Standard methods for the examination of water and wastewater. 17th ed. APHA, AWWA, WPCF. Washington, D.C., USA. 3. Dionisi, H.M., A.C. Layton, G. Harms, I.R. Gregory, K.G. Robinson, and G.S. Sayler Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR. Appl. Environ. Microbiol. 68, Hiorns, W.D., R.C. Hastings, I.M. Head, A.J. McCarthy, J.R. Saunders, R.W. Pickup, and G.H. Hall Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment. Microbiology 141, Koops, H.P., B. Bötcher, U.C. Möller, A. Pommerening-Röser, and G. Stehr Classification of eight new species of ammoniaoxidizing bacteria: Nitrosomonas communis sp. nov., Nitrosomonas urea sp. nov., Nitrosomonas aestuarii sp. nov., Nitrosomonas marina sp. nov., Nitrosomonas nitrosa sp. nov., Nitrosomonas eutropha sp. nov., Nitrosomonas oligotropha sp. nov. and Nitrosomonas halophila sp. nov. J. Gen. Microbiol. 137, Koops, H.P. and A. Pommerening-Röser Distribution and ecophysiology of the nitrifying bacteria emphasizing cultured species. FEMS Microbiol. Ecol. 37, Kowalchuk, G.A. and J.R. Stephen Ammonia-oxidizing bacteria: A model for molecular microbial ecology. Ann. Rev. Microbiol. 55, Limpiyakorn, T., F. Kurisu, and O. Yagi Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance. Wat. Sci. Technol. 54, Limpiyakorn, T., Y. Shinohara, F. Kurisu, and O. Yagi Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo. FEMS Microbiol. Ecol. 54, McCune, B. and J.B. Grace Analysis of ecological communities. Gleneden Beach, OR, MJM Software Design. 11. Metcalf and Eddy Wastewater engineering: treatment and reuse. 4th ed. New York, NY, McGraw-Hill. 12. Park, H.D., S.Y. Lee, and S. Hwang Redundancy analysis demonstrated the relevance of temperature to ammonia-oxidizing bacterial community compositions in a full-scale nitrifying biore-

7 118 Hee-Deung Park Kor. J. Microbiol actor treating saline wastewater J. Microbiol. Biotechnol. in print. 13. Park, H.-D., J.M. Regan, and D.R. Noguera Molecular analysis of ammonia-oxidizing bacterial populations in aerated-anoxic Orbal processes. Wat. Sci. Technol. 46, Park, H.D., L.M. Whang, S.R. Reusser, and D.R. Noguera Taking advantage of aerated-anoxic operation in a full-scale University of Cape Town (UCT) process. Wat. Environ. Res. 78, Purkhold, U., A. Pommerening-Roser, S. Juretschko, M.C. Schmid, H.-P. Koops, and M. Wagner Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rrna and amoa sequence analysis: implications for molecular diversity surveys. Appl. Environ. Microbiol. 66, Rotthauwe, J.H., K.P. Witzel, and W. Liesack The ammonia monooxygenase structural gene amoa as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63, Saitou, N. and M. Nei The neighbor-joining method - a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, ter Braak, C.J.F Canonical correspondence analysis: a new eigenvector technique for multivariate direct gradient analysis. Ecology 67, Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, US-EPA Manual: Nitrogen control. Cincinnati, OH, US- EPA. 21. Wagner, M., D.R. Noguera, S. Juretschko, G. Rath, H.P. Koops, and K.H. Schleifer Combining fluorescent in situ hybridization (FISH) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge. Wat. Sci. Technol. 37, Watson, S.W., E. Bock, H. Harms, H.-P. Koops, and A.B. Hooper Nitrifying bacteria, pp In J.T. Staley, M.P. Bryant, N. Pfenning, and J.G. Holts (ed.), Bergey's Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore, MD, USA. (Received March 31, 2009/Accepted April 21, 2009) ABSTRACT : Characterization and Composition of Ammonia-Oxidizing Bacterial Community in Full- Scale Wastewater Treatment Bioreactors Hee-Deung Park (School of Civil, Environmental and Architectural Engineering, Korea University, Seoul , Republic of Korea) Ammonia-oxidizing bacteria (AOB) are chemolithoautotrophs that play a key role in nitrogen removal from advanced wastewater treatment processes. Various AOB species inhabit and their community compositions vary over time in the wastewater treatment bioreactors. In this study, a hypothesis that operational and environmental conditions affect both the community compositions and the diversity of AOB in the bioreactors was proposed. To verify the hypothesis, the clone libraries based on ammonia monooxygenase subunit A were constructed using activated sludge samples from aerobic bioreactors at the Pohang, the Palo Alto, the Nine Springs, and the Marshall wastewater treatment plants (WWTPs). In those bioreactors, AOB within the Nitrosomonas europaea, N. oligotropha, N.-like, and Nitrosospira lineages were commonly found, while AOB within the N. communis, N. marina, and N. cryotolerans lineages were rarely detected in the samples. The AOB community structures were different in the bioreactors: AOB within the N. oligotropha lineage were the major microorganisms in the Pohang, the Palo Alto, and the Marshall WWTPs, while AOB within the N. europaea lineage were dominant in the Nine Springs WWTP. The correlations between the AOB community compositions of the wastewater treatment bioreactors and their operational (HRT, SRT, and MLSS) and environmental conditions (temperature, ph, COD, NH 3, and NO 3 - ) were evaluated using a multivariate statistical analysis called the Redundancy Analysis (RDA). As a result, COD and NO 3 - concentrations in the bioreactors were the statistically significant variables influencing the AOB community structures in the wastewater treatment bioreactors.

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82-01.fm w y wz 8«( 2y) 57~61, 2005 J. of the Korean Society for Environmental Analysis p w w Á Á w w» y l Analysis of Influence Factors and Corrosion Characteristics of Water-pipe in Potable Water System Jae Seong