The Korean Journal of Microbiology, Vol. 42, No. 1, March 2006, p. 26-33 Copyright 2006, The Microbiological Society of Korea Biological Nitrogen Removal System ³ ½ Á 1 Á z* w w w y œw w (Biological nitrogen removal; BNR) l z œ ww» w y ü ³ 16S rrna PCR terminal restriction fragment length polymorphism (T- RFLP) w w. w BNR l ü š w š l, Nutrient Removal Laboratory l,» w Sequencing Batch Reactor (SBR) l š, x y ³ β-proteobacteria w r y w. y ³ r» w SBR œ Nitrosomonas Nitrosolobus w y w. ù œ β-proteobacteria w ³ Cardococcus australiensis» ƒ w. w, y ³ w, SBR œ y ³ y ƒ z ùkû. w ƒ BNR l w s ƒ wš ³ x wš w. Key words ý 16S rrna, ammonia-oxidizing bacteria, biological nitrogen removal, T-RFLP s š, v w, œ vw» j (3), wì y w (algae) g w y ƒ y j. y (nitrification) k (denitrification) w w (Biological Nitrogen Removal; BNR) s œ w» (22) z (14), ƒ y y y s œ (9). y Nitrosomonas y ³ Nitrobacter y ³ w. y» (NH 4 +, NO 2, NO 3 ) ƒ xk y g j w k» s w w w (8). y ³ ¼ û w (19) sƒ sƒ» m w w» (11)., w y w š. w w w, 16S rrna y w ƒ w š. 16S rrna ³ w š,» y q w w (2). w Ribosomal Database Project (RDP; http://rdp.cme.msu.edu) œw *To whom correspondence should be addressed. Tel: 043-261-3261, Fax: 043-264-9600 E-mail: donghun@chungbuk.ac.kr 16S rrna database ƒ»» p ³ w, w PCR primer w ke ƒ ƒ w (5). 16S rrna terminal restriction fragment length polymorphism (T-RFLP) w z w r r w w, wù terminal(5' 3') restriction fragment (T-RF)ƒ p tw. w y (1), m (6), (18) w k w ƒ w (13, 15). ü w š œ œ xy, ³ w š œ š, ³ k Sequencing Batch Reactor (SBR) š š, y w œ š. A/Où A 2 /O w œ œ y, ù w œ š ( ƒy» l; http://www.konetic.or. kr). ü š BNR l w š l, Nutrient Removal Laboratory (NRL) l,» w SBR l y ³ T- RFLP w wš, BNR l z œ w ³ w œwš w. 26
Vol. 42, No. 1 BNR system ³ 27 w x ³ w 3 Biological Nitrogen Removal (BNR) l, w š l, NRL l, SBR l l y ù ³ w (Fig. 1). ƒ BNR l s w s,» w y z w. w w w» w Miler (17) bead beating xw w. (14,000 g, 10, 4 C) o 0.1 mm zirconia glass bead (Biospec, USA) 0.5 g, phosphate buffer (100 mm NaH 2 PO 4, ph 8.0) 300 µl, SDS solution (10%[wt/vol] SDS, 100 mm NaCl, 500 mm Tris-HCl, ph 8.0) 300 µl, chloroform: isoamylalcohol (24:1) 300 µl š, microtube mixer (TOMY SEIKO, Japan) w 5 bead beating w. (14,000 g, 15, 4 o C) w z, d w phenol: chloroform:isoamylalcohol (25:24:1) chloroform:isoamylalcohol (24:1) ƒ 1z w. d cold isopropanol sodium acetate ( 0.3 M) ƒwš -20 C 2 ew w o e g. (14,000 g, 30, 4 o C) w z, k wš g w e. w TE (10 mm Tris-HCl, 1 mm EDTA, ph 8.0) z, 0.8% agarose gel» w y w Ultraclean TM DNA Purification kit (MO BIO, USA) w -20 C o w. T-RFLP w w s 16S rrna s eubacterial primer 27F (E. coli numbering 8-27 : 5'-AGAGTTTGATCMTGGCTCAG-3') 785R (E. coli numbering 785-804 : 5'-ACT ACCRGGGTATCT AATCC-3') w (7, 12). w Ammonia-oxidizing bacteria (AOB) 16S rrna s specific primer 27F Nso1225R (E.coli numbering 1225-1244 : 5'- CGCCATTGTATTACGTGTGA-3') w (4). T-RF w 27F biotinylated primer (27FB) w. PCR 1X (100 mm Tris-HCl, 400 mm KCl, 1.5 mm MgCl 2, 500 µg/ml BSA, ph 8.3), 160 µm dntps, 0.3 µm primer, x w (10-100 ng/µl) 1.5 unit Taq polymerase ƒw 50 µl yw. PCR 95 C 3» w o z, 95 C o 30, 785R primer 58 o C, Nso1225R primer 52 C o 30, 72 C o 1 30z wš, 72 C 10 w z o g. PCR product 0.8% agarose gel» w y w, Ultraclean TM DNA purification kit (MO BIO) z -20 C o w. Fig. 1. Scheme of Biological Nitrogen Removal (BNR) systems. (A) An advanced treatment system with plotting media; (B) Nutrient Removal Laboratory (NRL) system; (C) Operating strategy in the rumination type Sequencing Batch Reactor (SBR) system. Check marks indicate the sampling sites of nitrification reactors. 16S rdna T-RFLP PCR wz HaeIII, HhaI (TaKaRa, Japan) 5 unit ƒƒ ƒw 37 C 5 o g. wz w DNA 0.5 SSC (75 mm NaCl, 7.5 mm sodium citrate, ph 7.3) 3z w streptavidin paramagnetic particle (Promega, USA) 1 SSC ƒw 10 g. Magnetic stand streptavidin paramagnetic particle 0.1 SSC 4z z,
28 Kyung-Mi Kim et al. Kor. J. Microbiol 0.2 N NaOH ƒw 5 jš, 0.2 N NaOH ƒw 2 k biotin single-stranded DNA T-RF (sst-rf) z w. 25% NH 4 OH š 65 o C 10 g streptavidin l DNA g. (14,000 g, 4, 4 C)w d w o z, 20 œ w w. T-RF profile 6% polyacrylamide gel» w y w. 3µl loading dye buffer (95% formamide, 10 mm NaOH, 20 mm EDTA, 0.02% bromophenol blue, 0.02% xylene cyanol FF) 1.5 µl 3 95 C k o z, x. 1 TBE (90 mm Tris-borate, 2 mm EDTA, ph 8.0) 6% polyacrylamide gel (acrylamide: bisacrylamide = 19:1, 0.7 M urea) 1,900 V HaeIII 3, HhaI 2» w.» óù 10% acetic acid 30 š w z, 3 3z wš, silver staining solution (0.1% AgNO 3, 0.055% form-aldehyde) 30 w. Developing reagent (3.0% Na 2 CO 3, 0.055% formaldehyde, Na 2 S 2 O 3 2.0 mg/l) z 10% acetic acid w jš 3 2z w. GelCompar II program (Applied Maths, Belgium) w ƒ T-RF pattern Pearson w š, UPGMA (21) xk dendrogram w. w T-RF Richness (S), Shannon-Weiner diversity index (H) w (6). y ³ T-RF» y ³ T-RF» w» w amplified fragment length polymorphism (AFLP) (24) w. w z HhaI (TaKaRa, Japan) w PCR 0.2 N NaOH w sst-rf w double-stranded DNA T-RF (dst-rf) z w. dst-rf HhaI-adapter ligationw z, 27FB adapter (A2) primer PCR sw. HhaI-adapter A1 primer (5'-CGATCGACAGTGTACTCTAGTC-3') A2 primer (5'-GAC TAGAGTACA CTGTCGATCGCG-3') ƒƒ 25 µm ƒ z, 95 C 10 o z, 20 w w. 2 PCR l streptavidin paramagnetic particle (Promega, USA) w sst-rf w z, 6% polyacrylamide gel (acrylamide :bisacrylamide = 19:1, 0.7 M urea, 1 TBE)» w y w. T-RF band ³» ü, ³ 3 š 30 C o 12 g. d x w 27F A2 primer s PCR pgem-t vector (Promega, USA) w cloning w. w j w z, plasmid DNA w BaseStation TM DNA Fragment Analyzers (MJ Research, USA)» w. ³ ƒ BNR l 16S rrna w T- RFLP w (Fig. 2). HaeIII w T-RF profile BNR l œm w 221 bp, w 264 bp T-RF w. w œ reactor 4 252 bp T-RFƒ. NRL l SBR l 219 bp T-RF w š, NRL l 201 bp T-RFƒ. 16S Fig. 2. PAGE and cluster analysis of HaeIII and HhaI T-RF profiles of the eubacterial communities in 3 BNR systems. NRL1S; Solution of NRL oxic tank 1, NRL2S; Solution of NRL oxic tank 2, NRL1M; Media of NRL oxic tank 1, NRL2M; Media of NRL oxic tank 2, R2M; Reactor 2 media of an advanced treatment system with plotting media, R4M; Reactor 4 media of an advanced treatment system with plotting media, R4S; Reactor 4 solution of an advanced treatment system with plotting media, R2S; Reactor 2 solution of an advanced treatment system with plotting media, SBRM; Media of Sequencing Batch Reactor oxic tank.
Vol. 42, No. 1 BNR system ³ 29 rrna» database w w 264 bp T-RF Nitrospira, Desulfotomaculum, Hydrogenobacter sw ƒ wš, 252 bp T-RF Acinetobacter, Chlorobium ƒ w, 221 bp T- RF Nitrosomonas, Azoarcus, Burkholderia sw β-proteobacteira ƒ w. 219 bp T-RF Nitrosospira, Aquaspirillum, Comamonas sw β- Proteobacteira Firmicutes ƒ w, 201 bp T-RF β-, γ-proteobacteria, Bacteroidetes, Planctomycetes Ÿ w ƒ w. HhaI w T-RFs profile BNR l 364~367 bp, 205 bp, 116 bp T-RF w. w l 197 bp T-RF, NRL l 565~568 bp T-RFƒ l w. NRL l 151 bp T-RFƒ. 565~568 bp T-RF β-, γ-proteobacteria, Firmicutes ƒ w, 364~367 bp T-RF β-proteobacteria, Actinobacteria ƒ w. 205 bp T-RF β-proteobacteria, Pseudomonas, Firmicutes, Bacteriodetesƒ sw w š, 197 bp T-RF Brevibacillus, Flavobacterium, Pirellula ƒ w. 151 bp T-RF Cardococcus, Frankia, Paenibacillus ƒ wš, 116 bp T-RF Lactobacillus ƒ w. HaeIII HhaI w œm ƒ w Nitrosomonas, Nitrosolobus y ³ Algaligenes, Aquaspirillum, Comamonas, Clostridium, Dechlorimonas, Pseudomonas, Rhodococcus, Thiobacillus. y w x wš. HaeIII HhaI w T-RF pattern w (cluster analysis) w, ƒ l (Fig. 2). p ƒƒ ù. w l ƒ 58.9%, 28.7% 21.1% wù x w. NRL l ƒ 55.4% 12.6% ƒ. ƒ l l û, mw ƒ BNR l w s ƒ wš ³ x wš, l ü y w. T-RF pattern w w (Table 1). T-RF (richness, S) HaeIII profile 21~32, HhaI profile 20~32 š, (Shannon-Weiner diversity index, H) HaeIII profile 3.72-4.84, HhaI profile 4.03-4.72 ùkû. HaeIII profile ƒ» ƒ, w l reactor 2 NRL l oxic tank 1 ƒƒ œ z ew reactor 4 oxic tank 2. HhaI profile HaeIII ƒ š, w l reactor 2 wš ƒ. ƒ ƒ NRL l oxic tank 2 š, ƒ û w l reactor 4. HaeIII HhaI s³ r w l ƒ f, reactor 2ƒ reactor 4. NRL l oxic tank 1 ƒ û, oxic tank 2. Table 1. Diversity statistics calculated from HaeIII and HhaI T-RF profiles of the BNR system samples Parameter Sample a HaeIII-S b HaeIII-H c HhaI-S b HhaI-H c Average-H R2S 32 4.84 23 4.31 4.58 R2M 27 4.39 22 4.11 4.25 R4S 29 4.67 20 4.03 4.35 R4M 21 3.99 24 4.29 4.14 NRL1S 23 4.16 21 4.09 4.13 NRL1M 24 3.98 30 4.65 4.32 NRL2S 21 4.08 26 4.38 4.23 NRL2M 22 3.72 32 4.72 4.22 SBRM 23 4.29 27 4.51 4.40 a Sampling site : R2S; Reactor 2 solution of an advanced treatment system with plotting media, R2M; Reactor 2 media of an advanced treatment system with plotting media, R4S; Reactor 4 solution of an advanced treatment system with plotting media, R4M; Reactor 4 media of an advanced treatment system with plotting media, NRL1S; Solution of NRL oxic tank 1, NRL1M; Media of NRL oxic tank 1, NRL2S; Solution of NRL oxic tank 2, NRL2M; Media of NRL oxic tank 2, SBRM; Media of Sequencing Batch Reactor oxic tank b Richness (S) = number of distinct T-RF in a profile c Shannon-Weiner diversity index(h) = -Σ(Pi)(log 2 Pi) Pi is the proportion of an individual peak height.
30 Kyung-Mi Kim et al. Kor. J. Microbiol w, SBR l l w ƒ ƒ, NRL l ƒ û. y ³ y ³ ³ w» w 27FB primer Nso1225R specific primer w (Fig. 3). T-RF pattern w 565~568 bp, 364~367 bp, 205 bp T-RF ³ y ³ mw w y j ³ ƒ. Actinobacteria ƒ w 442~446 bp T-RF Cardococcus, Frankia, Paenibacillus w ƒ w 151 bp T-RF NRL l. p 151 bp T-RF ³ w NRL l w. y ³ mw ƒ w w l, NRL l, SBR l ³ ƒƒ 19.03%, 21.48%, 23.85% w.» ƒ w T-RFLP w BNR l œm y 565~568 bp, 364~367 bp, 205 bp, NRL l 151 bp band gel w» w (Table 2). w l reactor 2 565 bp T-RF Aquaspirillum 94%, 364 bp T-RF Nitrosomonas 98%, 205 bp T-RF β- Proteobacteria uncultured eubacterium 94%. NRL l oxic tank 1 568 bp T-RF uncultured beta proteobacterium 93%, 365 bp T- RF uncultured bacterium 94%, 205 bp T-RF Aquaspirillum 84%, NRL l 151 bp T-RF Cardococcus 95%. SBR l 568 bp T-RF 367 bp T-RF Nitrosomonas sp. JL21 94%, 205 bp T-RF Nitrosolobus multiformis 100%.» y ³ T-RF BNR l SBR l w l Nitrosomonas, Nitrosolobus ƒ y. š T-RFLP w w» ¾ ³ š (23). Regan (20) nested PCR w T-RFLP y ³ Nitrosomonas Nitrosospira y ³ Nitrobacter Nitrospira wš z š šwš. BNR l y j w ³ w» w T-RFLP w., HaeIII HhaI w œm ƒ w Nitrosomonas, Nitrosolobus y ³ Algaligenes, Aquaspirillum, Comamonas, Clostridium, Dechlorimonas, Pseudomonas, Rhodococcus, Thiobacillus, Clostridium Pseudomonas wš β-proteobacteria w (Fig. 2). mw s ƒ wš w y y x ƒ l. w z ƒ f ³ ³ ƒ j» ƒ. Tsuneda (22) w DGGE w aerobic upflow fluidized bed (AUFB) y ³ w Nitrosomonas w ³ w š šw š, Kindaichi (10) y ù biofilm Nitrospira, Nitrosomonas w ³ ƒƒ 39% 25% wš. 27F Nso1225R primer w T-RFLP mw y ³ w (Fig. 3)., HhaI w ³ mw BNR l w 565~568 bp, 364~367 bp, 205 bp T-RF Table 2. Phylogenetic diversity of the nucleotide sequences of T-RFs identified by BLAST search Sample a Length of T-RF (bp) Closest Microorganism (accession number) Similarity(%) Phylum R2M-1 565 Aquaspirillum arcticum (AB074523) 94 β-proteobacteria R2M-2 364 Nitrosomonas sp. Is32 (AJ621027) 98 β-proteobacteria R2M-3 205 Uncultured eubacterium clone from the denitrifying reactor (AJ412627) 94 β-proteobacteria NRL1M-1 568 Uncultured beta proteobacterium clone from the river (AJ421928) 93 β-proteobacteria NRL1M-2 365 Uncultured bacterium clone from the groundwater (AY662045) 94 β-proteobacteria NRL1M-3 205 Aquaspirillum serpens (AB074518) 84 β-proteobacteria NRL1M-4 151 Cardococcus australiensis (AY007722) 95 β-proteobacteria SBRM-1 568 Nitrosomonas sp. JL21 (AB000700) 94 β-proteobacteria SBRM-2 367 Nitrosomonas sp. JL21 (AB000700) 98 β-proteobacteria SBRM-3 205 Nitrosolobus multiformis (L35509) 100 β-proteobacteria a Sample: R2M; Media of reactor 2 in advanced treatment system with plotting media, NRL1M; Media of oxic tank 1 in NRL system, SBRM; Media of oxic tank in Sequencing Batch Reactor system
Vol. 42, No. 1 BNR system ³ 31 Fig. 3. PAGE of the HhaI digested T-RFs of ammonia-oxidizing bacteria. Lane M; Size marker, 1-1; Reactor 2 solution of an advanced treatment system with plotting media (R2S), 1-2; Reactor 2 media of an advanced treatment system with plotting media (R2M), 1-3; Reactor 4 solution of an advanced treatment system with plotting media (R4S), 1-4; Reactor 4 media of an advanced treatment system with plotting media (R4M), 2-1; Solution of NRL oxic tank 1 (NRL1S), 2-2; Media of NRL oxic tank 1 media (NRL1M), 2-3; Solution of NRL oxic tank 2 (NRL2S), 2-4; Media of NRL oxic tank 2 (NRL2M), 3; Media of Sequencing Batch Reactor oxic tank (SBRM). Asterisks indicate lanes that nucleotide sequences of major bands were analyzed. y ³ œ. 442~ 446 bp 151 bp T-RF NRL l w, p 151 bp T-RF ³ ƒ y ³ NRL l. w l SBR l NRL l w š ƒ. y ³ ³ r, w l z ƒ ƒƒ 16.59%, 21.47%, NRL l z ƒ ƒƒ 20.46%, 22.50%, SBR l 23.85% w. z ƒ y ³ ƒw š, z y ³ y z w. Table 2 T-RF» w. SBR l y ³ w Nitorosomona sp. JL21, Nitrosolobus multiformis 94~100%. BNR l w l Nitorosomonas sp. Is32 w 364 bp T-RF wš y ³ β- Proteobacteria w ³ y w. ³ mw y ³ w 565~568 bp, 364~367 bp, 205 bp T-RF SBR l y ³ w ƒ w. w l 364 bp T-RF y ³ w š, NRL l w ƒ w T-RF. NRL l ƒ w 151 bp T-RF Cardococcus australiensis w. Maszenan (16) w Cardococcus australiensis y y w ³ t qyx y g š šwš. SBR l» š y ³ y, l β-proteobacteriaƒ y y ³ ƒ w. BNR l y ù l T-RFLP w ³ y ³ w. ³ mw ³ k vš w w, y ³» ƒ w T-RFLP ww yw. y ³ T-RF» mw j» T-RF ³ ³.» ƒ w T- RFLP w yw ƒ. Nso1225R primer w y ³ wš,» w, y ³ β-proteobacteria ³ y. Calvo (4) β- Proteobacteria w y ³ w» w Nso1225 FISH probe reverse primer w y ³ w w. DGGE mw y ³ y wš» w 13 band 6 β-proteobacteria y ³ w 1 β-proteobacteria k y ³, ù 6 y ³ β-proteobacteria w š šwš. w RDP mw Nso1225R pimer 374 ³ ww., 320 ³ y ³ Sporobacter 3 ³, Nitrobacter 1 ³, Methylobacillus 1 ³, unidenified β-proteobacteria 42 ³ y ³ ³ ww. y ³ yw w w specific primer v w š ƒ. 2004w w w w.
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Vol. 42, No. 1 BNR system ³ 33 ABSTRACT : Structure of Bacterial Communities in Biological Nitrogen Removal System Kyung-Mi Kim, Sang-Ill Lee 1, and Dong-Hun Lee* (Department of Microbiology and the Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea, 1 Department of Environmental Engineering, Chungbuk National University, Cheongju 361-763, Korea) To understand the efficient process of biological nitrogen removal (BNR) system, the structure of bacterial communities in nitrification reactors was analyzed using PCR and terminal restriction fragment length polymorphism (T-RFLP) methods. In this study, we used an advanced treatment system with plotting media, Nutrient Removal Laboratory system, or the rumination type sequencing batch reactor (SBR) system. The terminal restriction fragments of ammonia-oxidizing bacteria (AOB) and other β-proteobacteria were observed in all of three BNR systems. The nucleotide sequence analysis of terminal restriction fragments showed that Nitrosomonas and Nitrosolobus were major populations of AOB in SBR system, whereas uncultured β-proteobacteria and Cardococcus australiensis were the predominant groups in other two BNR systems. Also the SBR system may be more efficient to enrich AOB. These results indicate that the different structure of bacterial community may be developed depending on the wastewater treatment systems, although the same influent is used.